Homogeneous detection of a target through nucleic acid ligand-ligand beacon interaction

ABSTRACT

A method for detecting a target molecule in a test mixture suspected of containing said target molecule is described. The nucleic acid ligand capable of binding to the target molecule has a first sequence A and a second sequence B, which are partially complementary sequences that form an imperfect intramolecular duplex, which unwinds upon the binding of the target to the nucleic acid ligand. Sequences A and B are able to participate in extramolecular hybridization reactions only when the duplex is unwound. Three different cascade nucleic acids contain a first sequence and a second sequence, which are partially complementary sequences. At least one sequence is exactly complementary to A or B. The second sequence may be complementary to A or B, or may be a third sequence C, or its complement. The test mixture suspected of containing the target molecule is contacted with the nucleic acid ligand, causing the duplex of the nucleic acid ligand to unwind such that sequences A and B become available for extramolecular hybridization. This mixture is contacted with the first, second, and third cascade nucleic acids so that the unpaired A and B sequences triggers a cascade of intermolecular hybridization involving the cascade nucleic acids in which intermolecular hybridization takes place between A and its complement, B and its complement, and between C its complement, leading to the formation of a multimolecular hybridization complex. The presence of the multimolecular hybridization complex is then detected.

This application is a 371 of PCT/US98/26599, filed Dec. 15, 1998,entitled, “Homogeneous Detection of a Target Through Nucleic AcidLigand-Ligand Beacon Interaction.” PCT/US98/26599 claims the benefit ofU.S. Provisional Application Ser. No. 60/068,135, filed Dec. 15, 1997,entitled “System for Amplifying Flourescent Signal Through HybridizationCascade” and is a continuation in part of U.S. Application Ser. No.09/157,206, filed Sep. 18, 1998, entitled “Homogeneous Detection of aTarget Through Nucleic Acid Ligand-Ligand Beacon Interaction,” now U.S.Pat. No. 5,989,823.

FIELD OF THE INVENTION

This invention is directed to novel methods for the highly selectivedetection of specific target molecules. In one embodiment the methodsdescribed can be used to detect exceedingly low concentrations of saidtarget molecules by virtue of a highly efficient signal amplificationmechanism. In another embodiment the binding of a nucleic acid ligand toa target molecule is accompanied by a change in the fluorescencespectrum of the assay solution. The subject invention will be useful inany application where it is desired to detect a target molecule.

BACKGROUND OF THE INVENTION

Techniques that allow specific detection of target molecules or analytesare important for many areas of research, as well as for clinicaldiagnostics. Central to most detection techniques are ligands thatdictate specific and high affinity binding to a target molecule ofinterest. In immunodiagnostic assays antibodies mediate specific andhigh affinity binding, whereas in assays detecting nucleic acid targetsequences, complementary oligonucleotide probes fulfill this role. Todate, antibodies have been able to provide molecular recognition needsfor a wide variety of target molecules and have been the popular choiceof the class of ligands for developing diagnostic assays.

Recently, a novel class of oligonucleotide probes, referred to asmolecular beacons, that facilitate homogeneous detection of specificnucleic acid target sequences has been described (Piatek et al. (1998)Nature Biotechnol. 16:359-363; Tyagi and Kramer (1996) Nature Biotecnol.14:303-308). Molecular beacons are nucleic acid sequences that contain afluorophore and a quencher (FIG. 1; star and filled circle,respectively). By design, molecular beacons are expected to fold intostem-loop structures in which the fluorophore is placed in closeproximity to the quencher. When the molecular beacon is illuminated withlight corresponding to the excitation wavelength of the fluorescentgroup, no fluorescence is observed, because energy transfer occursbetween the fluorescent group and the quenching group, such that lightemitted from the fluorescent group upon excitation is absorbed by thequenching group.

The loop region of molecular beacons is designed to contain a nucleotidesequence complementary to the target sequence of interest. When themolecular beacon is contacted with sequences complementary to the loop,the loop hybridizes to this sequence. This process is energeticallyfavored as the intermolecular duplex formed is longer, and thereforemore stable, than the intramolecular duplex formed in the stem region.As this intermolecular double helix forms, torsional forces aregenerated that cause the stem region to unwind. As a result, thefluorescent group and the quenching group become spatially separatedsuch that the quenching group is no longer able to efficiently absorblight emitted from the fluorescent group. Thus, binding of the molecularbeacon to its target nucleic acid sequence is accompanied by an increasein fluorescence emission from the fluorescent group. Molecular beaconsundergo intermolecular hybridization upon interaction with the specifictarget sequence. Molecular beacons have been used for homogeneousdetection of specific nucleic acid sequences, both DNA and RNA (Leone etal. (1998) Nucleic Acids Research 26:2150-2155; Piatek et al. (1998)Nature Biotechnol. 16:359-363; Tyagi and Kramer (1996) Nature Biotecnol.14:303-308).

It is possible to simultaneously use two or more molecular beacons withdifferent sequence specificities in the same assay. In order to do this,each molecular beacon is labeled with at least a different fluorescentgroup. The assay is then monitored for the spectral changescharacteristic for the binding of each particular molecular beacon toits complementary sequence. In this way, molecular beacons have beenused to determine whether an individual is homozygous wild-type,homozygous mutant or heterozygous for a particular mutation. Forexample, using one quenched-fluorescein molecular beacon that recognizesthe wild-type sequence and another rhodamine-quenched molecular beaconthat recognizes a mutant allele, it is possible to genotype individualsfor the β-chemokine receptor (Kostrikis et al. (1998) Science279:1228-1229). The presence of only a fluorescein signal indicates thatthe individual is wild-type, and the presence of rhodamine signal onlyindicates that the individual is a homozygous mutant. The presence ofboth rhodamine and fluorescein signal is diagnostic of a heterozygote.Tyagi and coworkers have even described the simultaneous use of fourdifferently labeled molecular beacons for allele discrimination. (Tyagiet al. (1998) Nature Biotechnology 16:49-53).

Although useful for the detection of nucleic acid targets, molecularbeacons have not been used for detecting other types of molecules.Indeed, there has been no suggestion made in the art that molecularbeacons can be used for anything other than detecting specific nucleicacids in mixtures containing a plurality of nucleic acids. Detection ofnucleic acids is undeniably important, but in manyapplications—especially medical diagnostic scenarios—detection ofnon-nucleic acid molecules, such as proteins, sugars, and smallmetabolites, is required.

In general, the detection of non-nucleic acid target molecules is a morecomplicated matter than the detection of nucleic acids, and no singlemethod is universally applicable. Specific proteins may be detectedthrough the use of antibody-based assays, such as an enzyme linkedimmunoassay (ELISA). In one form of ELISA, a primary antibody binds tothe protein of interest, and signal amplification is achieved using alabeled secondary antibody that can bind to multiple sites on theprimary antibody. This technique can only be used to detect moleculesfor which specific antibodies exist. The generation of new antibodies isa time consuming and very expensive procedure and many proteins are notsufficiently immunogenic to generate antibodies in host animals.Furthermore, it is often necessary to measure and detect smallmolecules, such as hormones and sugars. that are generally not amenableto antibody recognition. In these cases, enzymatic assays for thespecific molecule are often required.

The discovery of the SELEX™ (Systematic Evolution of Ligands byEXponential enrichment) process enables the identification of nucleicacid-based ligands, referred to as aptamers, that recognize moleculesother than nucleic acids with high affinity and specificity (Ellingtonand Szostak (1990) Nature 346:818-822; Gold et al. (1995) Ann. Rev.Biochem. 64:763-797; Tuerk and Gold (1990) Science 249:505-510).Aptamers have been selected to recognize a broad range of targets,including small organic molecules as well as large proteins (Gold et al.(1995) Ann. Rev. Biochem. 64:763-797; Osborne and Ellington (1997) Chem.Rev. 97:349-370). In most cases, affinities and specificities ofaptamers to these targets are comparable or better than those ofantibodies. In contrast to antibodies whose identification andproduction completely rest on animals and/or cultured cells, both theidentification and production of aptamers takes place in vitro withoutany requirement for animals or cells. Aptamers are produced by solidphase chemical synthesis, an accurate and reproducible process withconsistency among production batches. Aptamers are stable to long-termstorage at room temperature. Moreover, once denatured, aptamers caneasily be renatured, a feature not shared by antibodies. These inherentcharacteristics of aptamers make them attractive for diagnosticapplications.

The SELEX process is a method for the in vitro evolution of nucleic acidmolecules with highly specific binding to target molecules and isdescribed in U.S. patent application Ser. No. 07/536,428. filed Jun. 11,1990, entitled “Systematic Evolution of Ligands by ExponentialEnrichment,” now abandoned; U.S. patent application Ser. No. 07/714,131,filed Jun. 10, 1991. entitled “Nucleic Acid Ligands,” now U.S. Pat. No.5,475,096; U.S. patent application Ser. No. 07/931,473, filed Aug. 17,1992, entitled “Methods for Identifying Nucleic Acid Ligands,” now U.S.Pat. No. 5,270,163 (see also, WO 91/19813), each of which isspecifically incorporated by reference herein in its entirety. Each ofthese applications, collectively referred to herein as the SELEX patentapplications, describes a fundamentally novel method for making anucleic acid ligand to any desired target molecule. The SELEX processprovides a class of products which are referred to as nucleic acidligands or aptamers, each ligand having a unique sequence, and which hasthe property of binding specifically to a desired target compound ormolecule. Each SELEX process-identified nucleic acid ligand is aspecific ligand of a given target compound or molecule. The SELEXprocess is based on the unique insight that nucleic acids havesufficient capacity for forming a variety of two- and three-dimensionalstructures and sufficient chemical versatility available within theirmonomers to act as ligands (form specific binding pairs) with virtuallyany chemical compound, whether monomeric or polymeric. Molecules of anysize or composition can serve as targets.

The SELEX method involves selection from a mixture of candidateoligonucleotides and step-wise iterations of binding, partitioning andamplification, using the same general selection scheme, to achievevirtually any desired criterion of binding affinity and selectivity.Starting from a mixture of nucleic acids, preferably comprising asegment of randomized sequence, the SELEX method includes steps ofcontacting the mixture with the target under conditions favorable forbinding, partitioning unbound nucleic acids from those nucleic acidswhich have bound specifically to target molecules, dissociating thenucleic acid-target complexes, amplifying the nucleic acids dissociatedfrom the nucleic acid-target complexes to yield a ligand-enrichedmixture of nucleic acids, then reiterating the steps of binding,partitioning, dissociating and amplifying through as many cycles asdesired to yield highly specific high affinity nucleic acid ligands tothe target molecule.

The basic SELEX method has been modified to achieve a number of specificobjectives. For example U.S. patent application Ser. No. 07/960,093,filed Oct. 14, 1992, entitled “Method for Selecting Nucleic Acids on theBasis of Structure,” abandoned in favor of U.S. patent application Ser.No. 08/198,670, now U.S. Pat. No. 5,707,796, describes the use of theSELEX process in conjunction with gel electrophoresis to select nucleicacid molecules with specific structural characteristics, such as bentDNA. U.S. patent application Ser. No. 08/123,935, filed Sep. 17, 1993,entitled “Photoselection of Nucleic Acid Ligands,” now abandoned (seeU.S. patent application Ser. No. 08/612,895, filed Mar. 8, 1996.entitled “Systematic Evolution of Ligands by Exponential Enrichment:Photoselection of Nucleic Acid Ligands and Solution SELEX, now U.S. Pat.No. 5,763,177), describes a SELEX process-based method for selectingnucleic acid ligands containing photoreactive groups capable of bindingand/or photocrosslinking to and/or photoinactivating a target molecule.U.S. patent application Ser. No. 08/134,028, filed Oct. 7. 1993,entitled “High-Affinity Nucleic Acid Ligands That Discriminate BetweenTheophylline and Caffeine,” abandoned in favor of U.S. patentapplication Ser. No. 08/443,957, now U.S. Pat. No. 5,580,737, describesa method for identifying highly specific nucleic acid ligands able todiscriminate between closely related molecules, which can benon-peptidic, termed Counter-SELEX. U.S. patent application Ser. No.08/143,564, filed Oct. 25, 1993, entitled “Systematic Evolution ofLigands by Exponential Enrichment: Solution SELEX,” abandoned in favorof U.S. patent application Ser. No. 08/461,069, now U.S. Pat. No.5,567,588, describes a SELEX process-based method which achieves highlyefficient partitioning between oligonucleotides having high and lowaffinity for a target molecule.

The SELEX method encompasses the identification of high-affinity nucleicacid ligands containing modified nucleotides conferring improvedcharacteristics on the ligand, such as improved in vivo stability orimproved delivery characteristics. Examples of such modificationsinclude chemical substitutions at the ribose and/or phosphate and/orbase positions. SELEX process-identified nucleic acid ligands containingmodified nucleotides are described in U.S. patent application Ser. No.08/117,991. filed Sep. 8, 1993. entitled “High Affinity Nucleic Acidligands Containing Modified Nucleotides,” abandoned in favor of U.S.patent application Ser. No. 08/430,709. now U.S. Pat. No. 5,660,985,that describes oligonucleotides containing nucleotide derivativeschemically modified at the 5- and 2′-positions of pyrimidines. U.S.patent application Ser. No. 08/134,028, supra, describes highly specificnucleic acid ligands containing one or more nucleotides modified with2′-amino (2′-NH₂). 2′-fluoro (2′-F), and/or 2′-O-methyl (2′-OMe). U.S.patent application Ser. No. 08/264,029, filed Jun. 22, 1994, entitled“Novel Method of Preparation of Known and Novel 2′ Modified Nucleosidesby Intramolecular Nucleophilic Displacement,” now abandoned, describesoligonucleotides containing various 2′-modified pyrimidines.

The SELEX method encompasses combining selected oligonucleotides withother selected oligonucleotides and non-oligonucleotide functional unitsas described in U.S. patent application Ser. No. 08/284,063, filed Aug.2, 1994, entitled “Systematic Evolution of Ligands by ExponentialEnrichment: Chimeric SELEX,” now U.S. Pat. No. 5,637,459 and U.S. patentapplication Ser. No. 08/234,997, filed Apr. 28, 1994, entitled“Systematic Evolution of Ligands by Exponential Enrichment: BlendedSELEX,” now U.S. Pat. No. 5,683,867, respectively. These applicationsallow the combination of the broad array of shapes and other properties,and the efficient amplification and replication properties ofoligonucleotides with the desirable properties of other molecules.

U.S. patent application Ser. No. 07/964,624, filed Oct. 21, 1992,entitled “Nucleic Acid Ligands to HIV-RT and HIV-1 Rev,” now U.S. Pat.No. 5,496,938, describes methods for obtaining improved nucleic acidligands after SELEX has been performed. U.S. patent application Ser. No.08/400,440, filed Mar. 8, 1995, entitled “Systematic Evolution ofLigands by Exponential Enrichment: Chemi-SELEX,” now U.S. Pat. No.5,705,337, describes methods for covalently linking a ligand to itstarget.

The SELEX method further encompasses combining selected nucleic acidligands with lipophilic compounds or non-immunogenic, high molecularweight compounds in a diagnostic or therapeutic complex as described inU.S. patent application Ser. No. 08/434,465, filed May 4, 1995, entitled“Nucleic Acid Ligand Complexes,” U.S. Pat. No. 6,011,020. VEGF nucleicacid ligands that are associated with a lipophilic compound, such asdiacyl glycerol or dialkyl glycerol, in a diagnostic or therapeuticcomplex are described in U.S. patent application Ser. No. 08/739,109,filed Oct. 25, 1996. entitled “Vascular Endothelial Growth Factor (VEGF)Nucleic Acid Ligand Complexes,” U.S. Pat. No. 5,859,228. VEGF nucleicacid ligands that are associated with a lipophilic compound, such as aglycerol lipid, or a non-immunogenic, high molecular weight compound,such as polyethylene glycol, are further described in U.S. patentapplication Ser. No. 08/897,35 1 filed Jul. 21, 1997, entitled “VascularEndothelial Growth Factor (VEGF) Nucleic Acid Ligand Complexes,” U.S.Pat. No. 6,051,698. VEGF nucleic acid ligands that are associated with anon-immunogenic, high molecular weight compound or lipophilic compoundare also further described in PCT/US97/18944, filed Oct. 17, 1997,entitled “Vascular Endothelial Growth Factor (VEGF) Nucleic Acid LigandComplexes.” Each of the above described patent applications whichdescribe modifications of the basic SELEX procedure are specificallyincorporated by reference herein in their entirety.

It is an object of the present invention to provide methods that can beused to detect virtually any non-nucleic acid target molecule in a testmixture, using nucleic acid reagents that are easily and cheaplymanufactured.

It is a further object of the instant invention to provide a method foradapting molecular beacons in order to detect non-nucleic acid targetmolecules in a test mixture.

Another object of the instant invention is to provide a single,universal assay for virtually any non-nucleic acid target molecule inwhich measurements of fluorescence emission are used to determine theconcentration of the target.

SUMMARY OF THE INVENTION

The present invention includes methods for detecting the binding ofnucleic acid ligands to their cognate target molecules. The methods relyon the insight that nucleic acid ligands can be recognized by molecularbeacons in a target-dependent context. The methods and reagentsdescribed herein allow, for the first time, virtually the detection ofvirtually any target molecule.

The invention uses novel molecular beacons, termed ligand beacons, thathybridize to nucleic acid ligands only under preselected conditions. Insome embodiments, the ligand beacon can only hybridize to nucleic acidligands that are free of their cognate target; in other embodiments, theligand beacon can only hybridize to nucleic acid ligands that are boundto their cognate targets. In either case, the binding of nucleic acidligand to target is accompanied by a measurable change in the spectralproperties of the ligand beacon. Conventional molecular beacons known inthe art are used to recognize complementary nucleic acid sequences,e.g., genomic sequences and sequences specific to pathogens. Bycontrast, ligand beacons recognize nucleic acid ligands with both aparticular sequence and a particular configuration. The configuration ofthe nucleic acid ligand changes when it is or is not bound to itscognate target.

In one embodiment the method for identifying the presence of a targetmolecule in a test mixture comprises: introducing a nucleic acid ligandto the target and a ligand beacon to the test mixture; wherein theligand beacon comprises: a) a nucleic acid sequence complementary to atleast a portion of the nucleic acid ligand, b) a fluorescent group, andc) a fluorescence-modifying group; wherein the emission profile of saidfluorescent group is different when said target molecule is present inthe test mixture from when said target molecule is not present; andmeasuring the fluorescence emission of said ligand beacon, whereby thepresence of said target molecule is determined.

The methods described herein provide, for the first time, a singleuniversal method for target molecule detection which simply involvesanalyzing fluorescence emission. The reagents and methods describedherein are particularly suitable for diagnostic assays. Diagnosticassays that require quantitative measurements (e.g., measurements of ahormone or sugar level) are possible according to the present inventionby simply comparing the fluorescence measurement with that obtained froma control. Similarly, diagnostic assays requiring qualitative detectionof substances (e.g., the presence of a mutated gene product or thepresence of a pathogen) are also possible. The reagents can be used inassays for single substances or they can be used to simultaneouslymonitor a variety of substances in a single assay. Using differentfluorescent groups with spectroscopically resolvable emission spectra,this method allows for the simultaneous detection of multiple targets ina single vessel. In this homogeneous multiplexing approach, distinctfluorescent groups can be attached to different nucleic acid ligandsspecific to targets of interest.

In particular, the invention provides methods for performing assaysusing reagents attached to solid supports. In these embodiments, aplurality of nucleic acid ligands are attached to spatially discreteregions on solid supports, and contacted with the solution to beassayed. Using the detection methods described herein, measurements offluorescence at discrete sites on the solid support can reveal whetherparticular substances are present in the assay solution and in whatquantities. In this way, it is possible to assay for aplurality—potentially hundreds or even thousands—of different substancesin a single test. Arrays of nucleic acid ligands that can be used withthe methods and reagents described herein are detailed in U.S. patentapplication Ser. No. 08/990,436, filed Dec. 15, 1997, entitled “NucleicAcid Ligand Diagnostic Biochip,” which is incorporated herein byreference in its entirety.

The ligand beacon assay described here has several advantages. It is ahomogeneous assay that can be performed in plasma. It is a generalmethod to detect virtually any class of target molecule to which highaffinity and specific nucleic acid ligand is available. It consists ofthree simple steps:—addition of a nucleic acid ligand/aptamer, additionof a ligand beacon and measurement of fluorescence. It is fast, requiresless than 30 minutes, and amenable for high throughput screening. Sincemolecular beacons equipped with distinct fluorophores that emit atdifferent wavelengths have been used to detect more than one targetnucleic acid sequences in a single sample (Kostrikis et al. (1998)Science 279:1228-1229; Tyagi et al. (1998) Nature Biotechnol. 16:49-53),the ligand beacon assay is also amenable for multiplexing. In somerespects, this assay is quite analogous to fluorescence polarizationcompetitive immunoassay that is currently used in the clinical routine(Wilson et al. (1998) Clin. Chem. 44:86-91).

The present invention also includes methods for the detection of targetmolecules in test mixtures through the use of a hybridization cascadeinvolving a set of three or more mutually complementaryoligonucleotides. In this method, a first nucleic acid binds to a targetmolecule and the nucleic acid undergoes a conformational change thatexposes sequences to which other nucleic acids in the set can hybridize.The nucleic acids that hybridize to the first nucleic acid also undergoa conformational change during hybridization that similarly exposessequences to which other nucleic acids in the set can hybridize. Thesequences of the set of nucleic acids involved are chosen so that acascade of hybridization can occur between the members of the set. Thischain reaction of conformational change and hybridization will continueuntil one of the participating nucleic acids is depleted. Any nucleicacid structure that undergoes a hybridization-promoting conformationalchange upon (i) binding to a target molecule, and/or (ii) hybridizing toanother nucleic acid, is contemplated in the subject methods.

In a preferred embodiment, a set of at least three single-strandednucleic acids are used, wherein each nucleic acid has a domain with anintramolecular double helix. The sequences of the nucleic acids arechosen so that the regions that form the intramolecular helix in onemember of the set will hybridize more stably to those regions of anothermember of the set than to one another. For example, the set couldcomprise sequences as follows wherein the letters A, B and C signify aunique sequence in the intramolecular helical region, “/” signifiesimperfect intramolecular base pairing and “i” signifies a complementarysequence: (i) A/B, (ii) B′/C, (iii) C′/A′. Thus, if the first nucleicacid is A/B and these sequences become available for intermolecularbase-pairing upon target molecule binding, then the A segment will thenbind to the A′ segment of C′/A′, and the B segment will bind to the B′segment of B′/C. This in turn allows the C and C′ portions of the newlybound nucleic acids to bind to their complementary sequences in C′/A′and B′/C respectively. These reactions occur because intermolecularhelix formation is more energetically favored than intramolecular helixformation. This cascade of intermolecular helix formation between thethree members of the set results in the formation of a multimolecularhybridization complex.

The cascade of hybridization described is triggered by theconformational change of the first nucleic acid upon binding to thetarget molecule. In the case of nucleic acids with an intramoleculardouble helix, this conformational change is the dissolution of theintramolecular helix of the first nucleic acid. This exposes theantiparallel strands that comprise the double helix, and allows them toparticipate in hybridization reactions. This disruption will occur whenthe target molecule binding region of the nucleic acid binds to aspecific target molecule.

In some embodiments, the target-binding region of the first nucleic acidwill hybridize through Watson-Crick interactions to a target nucleicacid with sequence complementary to at least part of the loop region.Hence, binding of a single molecule of the first nucleic acid to asingle target nucleic acid will initiate the formation of themultimolecular complex described above.

In other embodiments, the target-binding region is a nucleic acid ligandcomprised of sequences that can bind to a non-nucleic acid targetmolecules through non-Watson-Crick interactions. Binding to a targetmolecule will bring about the same cascade of intermolecularhybridizations as described above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the use of molecular beacons, wherein the star representsa fluorescent group and the filled circle represents a quenching group.Separation of the quenching group from the fluorescent group uponbinding of the nucleic acid leads to the production of a fluorescentsignal.

FIGS. 2A-C illustrate schematically the principle behind the ligandbeacon assay.

FIG. 2A illustrates the interaction of an aptamer (nucleic acid ligand)with a molecular beacon (ligand beacon) whose nucleotide sequence in theloop is complementary to a nucleotide stretch in the aptamer. Thisinteraction causes the spatial separation of the fluorophore (star) fromthe quencher (pentagon) producing a fluorescence signal.

In FIG. 2B the aptamer-specific target protein binds to the aptamer in aspecific and high affinity manner, thereby protecting the aptamer frominteraction with the ligand beacon. When the aptamer-specific target ispresent in excess the addition of the ligand beacon would not generatefluorescence.

FIG. 2C depicts the expected change in fluorescence signal as a functionof the log of the concentration of the target. When the concentration ofthe target is exceedingly low, virtually all the aptamer molecules areavailable for generating high signal upon interacting with the ligandbeacon (upper box). When the target is in great excess to that of theaptamer, there is virtually no free aptamer available to generate signalupon hybridization to the ligand beacon (lower box). When the targetconcentration is not at these two extremes, the fluorescence signal isinversely proportional to the concentration of the target (middle box).

FIG. 3A depicts the primary and predicted secondary structures of Taqaptamer (SEQ ID NO:9), PDGF aptamer (SEQ ID NO:10), L-selectin aptamer(SEQ ID NO:11) and P-selectin aptamer (SEQ ID NO:12). P-selectin aptameris an RNA-based aptamer in which all pyrimidine nucleotides have 2′-Fsubstitutions on the sugar. All other aptamers are DNA-based sequences.Nucleotide stretches indicated in bold are complementary to the loopsequences in cognate ligand beacons shown in FIG. 3B.

FIG. 3B depicts the primary structures of the ligand beacons used in thestudy. These sequences are designed to fold into stem-loop structures inwhich five nucleotides (bold) in the termini form the stem, whereas thenucleotide stretch in the middle (italics) forms the loop. The 5′terminus of each ligand beacon carries the fluorophore, fluorescein (F),whereas the 3′ terminus contains DABCYL as the quencher (Q).

FIGS. 4A and B illustrate the interaction of DNA-based aptamer sequenceswith ligand beacons. The primary and predicted secondary structures ofthe aptamers and their corresponding ligand beacon sequences are shownin FIGS. 3A and B. The experimental procedure is described in Example 3.FIG. 4A depicts the results of the interaction of the PDGF aptamer withits ligand beacon. Closed circles indicate the signal generated by theligand beacon specific to the PDGF aptamer upon interaction with thePDGF aptamer with predicted 3-way junction structure. Open circlesrepresent the signal generated by the same ligand beacon when incubatedwith the truncated sequence (indicated in bold in FIG. 3A) without theextra nucleotides in the aptamer. Stars indicate the signal generated bythe same ligand beacon when mixed with the Taq aptamer. FIG. 4B depictsthe results of the interaction of Taq aptamer with its ligand beacon.Closed circles indicate the signal generated by the ligand beaconspecific to the Taq aptamer upon interaction with the Taq aptamer withpredicted stem-loop structure. Open circles represent the signalgenerated by the same ligand beacon when incubated with the truncatedsequence (indicated in bold in FIG. 3A) without the extra nucleotides inthe aptamer. Stars indicate the signal generated by the same ligandbeacon when mixed with the PDGF aptamer.

FIG. 4C illustrates the extent to which the change in fluorescenceemission is dependent on the presence of a specific target. In thiscase, no change in fluorescence emission is observed when a nucleic acidligand to Taq polymerase and its cognate ligand beacon are mixed withPDGF.

FIG. 5A depicts the results of the ligand beacon assay for PDGF inbuffer (Example 5). FIG. 5B depicts the results of control experimentscarried out in the same buffer at the same temperature.

FIG. 6 depicts the results of the ligand beacon assay for Taq polymerasein buffer.

FIG. 7 depicts the results of the ligand beacon assay for humanL-selectin in buffer.

FIG. 7A illustrates the detection of low concentrations of L-selectinwith 200 nM each of L-selectin aptamer and L-selectin ligand beacon.

FIG. 7B illustrates the detection of high concentrations of L-selectinwith 800 nM each of L-selectin aptamer and L-selectin ligand beacon.

FIG. 8A illustrates the interaction of the RNA based P-selectin aptamersequence with its ligand beacon.

FIG. 8B depicts the results of the ligand beacon assay for humanP-selectin.

FIG. 8C illustrates the specificity of the assay with respect to thesimilar proteins L-selectin and P-selectin.

FIG. 9 depicts the results of ligand beacon assays for detectingproteins in plasma.

FIG. 9A illustrates the results of a ligand beacon assay for human PDGFAB-dimer in plasma,

FIG. 9B illustrates the results of a ligand beacon assay for humanL-selectin in plasma and

FIG. 9C illustrates the results of a ligand beacon assay for humanP-selectin in plasma.

FIG. 10 depicts the early stages of a hybridization cascade using astem-loop nucleic acid set.

FIG. 11 depicts the bidirectional propagation of the hybridizationcascade.

FIG. 12 illustrates how the hybridization cascade is accompanied by thegeneration of an increasingly large fluorescence signal.

FIG. 13 illustrates that a tri-molecular non-cascade hybridizationproduct will have no fluorescence activity.

FIGS. 14A-D illustrate an example of cascade hybridization using threedifferent stem-loop sequences.

DETAILED DESCRIPTION OF THE INVENTION

The present invention describes methods for the detection and/orquantitation of virtually any target molecule in a mixture. Theinvention uses novel molecular beacons, termed ligand beacons, thathybridize to nucleic acid ligands only under preselected conditions. Insome embodiments, the ligand beacon can only hybridize to nucleic acidligands that are free of their cognate target; in other embodiments, theligand beacon can only hybridize to nucleic acid ligands that are boundto their cognate targets. In either case, the binding of nucleic acidligand to target is accompanied by a measurable change in the propertiesof the ligand beacon.

In one embodiment the method for identifying the presence of a targetmolecule in a test mixture comprises: introducing a nucleic acid ligandto the target and a ligand beacon to the test mixture; wherein theligand beacon comprises: a) a nucleic acid sequence complementary to atleast a portion of the nucleic acid ligand, b) a fluorescent group, andc) a fluorescence-modifying group; wherein the emission profile of saidfluorescent group is different when said target molecule is present inthe test mixture from when said target molecule is not present; andmeasuring the fluorescence emission of said ligand beacon, whereby thepresence of said target molecule is determined. In this embodiment, thepresent invention provides a single universal method for target moleculedetection which simply involves analyzing fluorescence emission.

The reagents and methods described herein are particularly suitable fordiagnostic assays. Diagnostic assays that require quantitativemeasurements (e.g. measurements of a hormone or sugar level) arepossible according to the present invention by simply comparing thefluorescence measurement with that obtained from a control. Similarly.diagnostic assays requiring qualitative detection of substances (e.g.,the presence of a mutated gene product or the presence of a pathogen)are also possible. The reagents can be used in assays for singlesubstances or they can be used to simultaneously monitor a variety ofsubstances in a single assay. Using different fluorescent groups withspectroscopically resolvable emission spectra, this method allows forthe simultaneous detection of multiple targets in a single vessel. Inthis homogeneous multiplexing approach, distinct fluorescent groups canbe attached to different nucleic acid ligands specific to targets ofinterest.

In another embodiment, this invention provides methods for performingassays using reagents attached to solid supports. In this embodiment, aplurality of nucleic acid ligands are attached to spatially discreteregions on solid supports, and contacted with the solution to beassayed. Using the detection methods described herein, measurements offluorescence at discrete sites on the solid support can reveal whetherparticular substances are present in the assay solution and in whatquantities. In this way, it is possible to assay for aplurality—potentially hundreds or even thousands—of different substancesin a single test. Arrays of nucleic acid ligands that can be used withthe methods and reagents described herein are detailed in U.S. patentapplication Ser. No. 08/990,436, filed Dec. 15, 1997, entitled “NucleicAcid Ligand Diagnostic Biochip,” which is incorporated herein byreference in its entirety.

The ligand beacon assay described herein is a homogeneous assay that canbe performed in plasma, it is a general method to detect virtually anyclass of target molecule to which high affinity and specific nucleicacid ligand is available, it consists of three simple steps:—addition ofa nucleic acid ligand/aptamer, addition of a ligand beacon andmeasurement of fluorescence, it fast, requiring less than 30 minutes andis amenable for high throughput screening.

The present invention also includes methods for the detection of targetmolecules in test mixtures through the use of a hybridization cascadeinvolving a set of three or more mutually complementaryoligonucleotides. Cascade hybridization can be used to detectexceedingly low concentrations of the target molecule by virtue of ahighly efficient signal amplification mechanism.

Various terms are used herein to refer to aspects of the presentinvention. To aid in the clarification of the description of thecomponents of this invention, the following definitions are provided.

“Nucleic acid” means either DNA, RNA, single-stranded ordouble-stranded, and any chemical modifications thereof. Modificationsinclude, but are not limited to, those which provide other chemicalgroups that incorporate additional charge, polarizability, hydrogenbonding, electrostatic interaction, and fluxionality to the nucleic acidligand bases or to the nucleic acid ligand as a whole. Suchmodifications include, but are not limited to, 2′-position sugarmodifications, 5-position pyrimidine modifications, 8-position purinemodifications, modifications at exocyclic amines, substitution of4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbonemodifications, methylations, unusual base-pairing combinations such asthe isobases isocytidine and isoguanidine and the like. Modificationscan also include 3′ and 5′ modifications such as capping.

“Nucleic acid ligand” refers to a non-naturally occurring nucleic acidhaving a desirable action on a target. A desirable action includes, butis not limited to, binding of the target, catalytically changing thetarget, reacting with the target in a way which A modifies/alters thetarget or the functional activity of the target, covalently attaching tothe target as in a suicide inhibitor, facilitating the reaction betweenthe target and another molecule. In a preferred embodiment, the actionis specific binding affinity for a target molecule, such target moleculebeing a three dimensional chemical structure other than a polynucleotidethat binds to the nucleic acid ligand through a mechanism whichpredominantly depends on Watson/Crick base pairing or triple helixbinding, wherein the nucleic acid ligand is not a nucleic acid havingthe known physiological function of being bound by the target molecule.Nucleic acid ligands include nucleic acids that are identified from acandidate mixture of nucleic acids, said nucleic acid ligand being aligand of a given target, by the method comprising: a) contacting thecandidate mixture with the target, wherein nucleic acids having anincreased affinity to the target relative to the candidate mixture maybe partitioned from the remainder of the candidate mixture; b)partitioning the increased affinity nucleic acids from the remainder ofthe candidate mixture; and c) amplifying the increased affinity nucleicacids to yield a ligand-enriched mixture of nucleic acids (the SELEXprocess).

Nucleic acid ligand includes nucleic acid sequences that aresubstantially homologous to the nucleic acid ligands actually isolatedby the SELEX method. By substantially homologous it is meant a degree ofprimary sequence homology in excess of 70%, most preferably in excess of80%. In the past it has been shown that various nucleic acid ligands toa specific target with little or no primary homology may havesubstantially the same ability to bind the target. For this reason, thisinvention also includes nucleic acid ligands that have substantially thesame ability to bind a target as the nucleic acid ligands identified bythe SELEX process. Substantially the same ability to bind a target meansthat the affinity is within a few orders of magnitude of the affinity ofthe ligands described herein. It is well within the skill of those ofordinary skill in the art to determine whether a givensequence—substantially homologous to those specifically describedherein—has substantially the same ability to bind a target.

The term “aptamer” is used interchangeably with the term “nucleic acidligand.”

“Target” means any compound or molecule of interest for which adiagnostic test is desired and where a nucleic acid ligand is known orcan be identified. A target can be a protein, peptide, nucleic acid,lipid, carbohydrate, polysaccharide, glycoprotein, hormone, receptor,antigen, antibody, virus, pathogen, toxic substance, substrate,metabolite, transition state analog, cofactor, inhibitor, drug, dye,nutrient, growth factor, cell, tissue, etc. without limitation. A targetcan also be modified in certain ways to enhance the likelihood of aninteraction between the target and the nucleic acid.

“Candidate mixture” refers to a mixture of nucleic acids of differingsequence from which to select a desired ligand. The source of acandidate mixture can be from naturally-occurring nucleic acids orfragments thereof, chemically synthesized nucleic acids, enzymaticallysynthesized nucleic acids or nucleic acids made by a combination of theforegoing techniques. In a preferred embodiment, each nucleic acid hasfixed sequences surrounding a randomized region to facilitate theamplification process.

The “SELEX™” methodology involves the combination of selection ofnucleic acid ligands which interact with a target in a desirable manner,for example binding to a protein, with amplification of those selectednucleic acids. Optional iterative cycling of the selection/amplificationsteps allows selection of one or a small number of nucleic acids whichinteract most strongly with the target from a pool which contains a verylarge number of nucleic acids. Cycling of the selection/amplificationprocedure is continued until a selected goal is achieved. The SELEXmethodology is described in the SELEX patent applications.

“Cascade hybridization” as defined herein refers to a process wherebythe binding of a nucleic acid to a target molecule promotes theformation of a multimolecular complex comprising several differentmutually complementary nucleic acids associated with one another throughWatson-Crick interactions.

“Energy transfer” refers to a process whereby an energy donatingchemical group upon excitation with light of a particular wavelengthyields a photon of a specific wavelength, whereupon said photon iscaptured and absorbed by an energy accepting group before said can bespectroscopically detected.

“Solid support” refers to any of a number of supports to which moleculesmay be attached through either covalent or non-covalent bonds. The solidsupports can be beads, filters or microfabricated surfaces, such asbiochips. Solid supports are typically made of inert materials that arefunctionalized on their surface to allow for the attachment of nucleicacids. This includes, but is not limited to, Langmuir-Bodgett films,functionalized glass, membranes, charged paper, nylon, germanium,silicon, PTFE, polystyrene, gallium arsenide, gold and silver. Any othermaterial known in the art that is capable of having functional groupssuch as amino, carboxyl, thiol or hydroxyl, incorporated on its surfaceis contemplated. This includes surfaces with any topology, suchspherical surfaces and grooved surfaces.

“Bodily fluid” refers to a mixture of molecules obtained from anorganism. Bodily fluids include, but are not limited to, whole blood,blood plasma, urine, semen, saliva, lymph fluid, meningal fluid,amniotic fluid, glandular fluid, sputum and cerebrospinal fluid. Bodilyfluid also includes experimentally separated fractions of all of thepreceding and solutions or mixtures containing homogenized solidmaterial, such as feces, tissues and biopsy samples.

“Test mixture” refers to any sample that contains a plurality ofmolecules. This includes, but is not limited to, bodily fluids asdefined above and any sample for environmental and toxicology testingsuch as contaminated water and industrial effluent.

“Fluorescent group” refers to a molecule that, when excited with lighthaving a selected wavelength, emits light of a different wavelength.Fluorescent groups include, but are not limited to, fluorescein,tetramethylrhodamine, Texas Red. BODIPY,5-[(2-aminoethyl)amino]napthalene-1-sulfonic acid (EDANS), coumarin andLucifer yellow. Fluorescent groups may also be referred to as“fluorophores”.

“Fluorescence-modifying group” refers to a molecule that can alter inany way the fluorescence emission from a fluorescent group. Afluorescence-modifying group generally accomplishes this through anenergy transfer mechanism. Depending on the identity of thefluorescence-modifying group, the fluorescence emission can undergo anumber of alterations, including, but not limited to, attenuation,complete quenching, enhancement, a shift in wavelength, a shift inpolarity, a change in fluorescence lifetime. One example of afluorescence-modifying group is a quenching group.

“Energy transfer” refers to the process by which the fluorescenceemission of a fluorescent group is altered by a fluorescence-modifyinggroup. If the fluorescence-modifying group is a quenching group, thenthe fluorescence emission from the fluorescent group is attenuated(quenched). Energy transfer can occur through fluorescence resonanceenergy transfer or through direct energy transfer. The exact energytransfer mechanisms in these two cases are different. It is to beunderstood that any reference to energy transfer in the instantapplication encompasses all of these mechanistically-distinct phenomena.

“Energy transfer pair” is used to refer to a group of molecules thatform a single complex within which energy transfer occurs. Suchcomplexes may comprise, for example, two fluorescent groups which may bedifferent from one another and one quenching group, two quenching groupsand one fluorescent group, or multiple fluorescent groups and multiplequenching groups. In cases where there are multiple fluorescent groupsand/or multiple quenching groups, the individual groups may be differentfrom one another e.g., one complex contemplated herein comprisesfluorescein and EDANS as fluorescent groups, and DABCYL as a quenchingagent.

“Quenching group” refers to any fluorescence-modifying group that canattenuate at least partly the light emitted by a fluorescent group. Werefer herein to this attenuation as “quenching.” Hence, illumination ofthe fluorescent group in the presence of the quenching group leads to anemission signal that is less intense than expected, or even completelyabsent. Quenching occurs through energy transfer between the fluorescentgroup and the quenching group. The preferred quenching group of theinvention is (4-dimethylaminophenylazo)benzoic acid (DABCYL).

“Fluorescence resonance energy transfer” or “FRET” refers to an energytransfer phenomenon in which the light emitted by the excitedfluorescent group is absorbed at least partially by afluorescence-modifying group. If the fluorescence-modifying group is aquenching group, then that group can either radiate the absorbed lightas light of a different wavelength or it can dissipate it as heat. FRETdepends on an overlap between the emission spectrum of the fluorescentgroup and the absorption spectrum of the quenching group. FRET alsodepends on the distance between the quenching group and the fluorescentgroup. Above a certain critical distance, the quenching group is unableto absorb the light emitted by the fluorescent group or can do so onlypoorly.

“Direct energy transfer” refers to an energy transfer mechanism in whichpassage of a photon between the fluorescent group and thefluorescence-modifying group does not occur. Without being bound by asingle mechanism, it is believed that in direct energy transfer, thefluorescent group and the fluorescence-modifying group interfere witheach others electronic structure. If the fluorescence-modifying group isa quenching group, this will result in the quenching group preventingthe fluorescent group from even emitting light. Quenching groups andfluorescent groups are frequently close enough together in the stem ofligand beacons that direct energy transfer can take place. For example,when DABCYL is located on one terminus of a ligand beacon, thisquenching group can efficiently quench almost all fluorescent groups onthe other terminus through direct energy transfer.

In general, quenching by direct energy transfer is more efficient thanquenching by FRET. Indeed, some quenching groups that do not quenchparticular fluorescent groups by FRET (because they do not have thenecessary spectral overlap with the fluorescent group) can do soefficiently by direct energy transfer. Furthermore, some fluorescentgroups can act as quenching groups themselves if they are close enoughto other fluorescent groups to cause direct energy transfer. Forexample, under these conditions, two adjacent fluorescein groups canquench one another's fluorescence effectively. For these reasons, thereis no limitation on the nature of the fluorescent groups and quenchinggroups useful for the practice of this invention.

“Ligand beacon” refers to a nucleic acid molecule, labeled with anenergy transfer pair, that can specifically hybridize to a nucleic acidligand under preselected conditions. Upon doing so, the ligand beaconundergoes a conformational change that causes the members of the energytransfer pair to move relative to one another such that the emissionfrom the fluorescent group is modified. Preferred energy transfer pairscomprise a fluorescent group and a quenching group. In preferredembodiments, the ligand beacon comprises a unimolecular stem-loopnucleic acid, wherein the fluorescent group and the quenching group areat the termini of the nucleic acid, and the loop comprises sequencesthat are at least partially complementary to sequences within thenucleic acid ligand. In some embodiments, the ligand beacon can onlyhybridize to the nucleic acid ligand when the nucleic acid ligand is notbound to its target. In other embodiments, the ligand beacon can onlyhybridize when the nucleic acid ligand is bound to its cognate target.In either case, hybridization of the ligand beacon to the nucleic acidligand is accompanied by a change in the fluorescence emission intensityof the ligand beacon.

Although the ligand beacon comprises a unimolecular stem-loop nucleicacid in preferred embodiments, there is no limitation on the structureof the ligand beacon. Any nucleic acid that can hybridize to a nucleicacid ligand, and in doing so undergo a conformational change that altersthe distance between nucleotides, is contemplated in the instantinvention. For example, nucleic acids configured as G-quartets may beuseful in this invention. These nucleic acid structures are formed byhydrogen bonding between the Hoogsteen and Watson-Crick faces of fourspatially adjacent guanosines. Adjacent quartets can stack on top of oneanother to form a highly symmetric and regular complex. Similarly,ligand beacons that undergo conformational changes in which initiallyseparated nucleotide positions become adjacent upon hybridizing tonucleic acid ligands are also included in the invention. These latterligand beacons, when labeled with fluorescent groups and quenchinggroups at the appropriate nucleotide positions undergo a decrease influorescence intensity upon binding to the nucleic acid ligand.

In a preferred embodiment, the nucleic acid ligands of the presentinvention are derived using the SELEX process, which is described inU.S. patent application Ser. No. 07/536,428, filed Jun. 11, 1990,entitled “Systematic Evolution of Ligands by EXponential Enrichrnent,”now abandoned, and U.S. patent application Ser. No. 07/714,131, filedJun. 10, 1991, entitled “Nucleic Acid Ligands,” now U.S. Pat.No.5,475,096, U.S. patent application Ser. No. 07/931,473, filed Aug.17, 1992, entitled “Methods for Identifying Nucleic Acid Ligands,” nowU.S. Pat. No. 5,270,163 (see also, WO 91/19813). These applications,each specifically incorporated herein by reference, are collectivelycalled the SELEX patent applications.

The SELEX process provides a class of products which are nucleic acidmolecules, each having a unique sequence, and each of which has theproperty of binding specifically to a desired target compound ormolecule. Target molecules are preferably proteins, but can also includeamong others carbohydrates, peptidoglycans and a variety of smallmolecules. SELEX methodology can also be used to target biologicalstructures, such as cell surfaces or viruses, through specificinteraction with a molecule that is an integral part of that biologicalstructure.

In its most basic form, the SELEX process may be defined by thefollowing series of steps:

1) A candidate mixture of nucleic acids of differing sequence isprepared. The candidate mixture generally includes regions of fixedsequences (i.e., each of the members of the candidate mixture containsthe same sequences in the same location) and regions of randomizedsequences. The fixed sequence regions are selected either: (a) to assistin the amplification steps described below, (b) to mimic a sequenceknown to bind to the target, or (c) to enhance the concentration of agiven structural arrangement of the nucleic acids in the candidatemixture. The randomized sequences can be totally randomized (i.e., theprobability of finding a base at any position being one in four) or onlypartially randomized (e.g., the probability of finding a base at anylocation can be selected at any level between 0 and 100 percent).

2) The candidate mixture is contacted with the selected target underconditions favorable for binding between the target and members of thecandidate mixture. Under these circumstances, the interaction betweenthe target and the nucleic acids of the candidate mixture can beconsidered as forming nucleic acid-target pairs between the target andthose nucleic acids having the strongest affinity for the target.

3) The nucleic acids with the highest affinity for the target arepartitioned from those nucleic acids with lesser affinity to the target.Because only an extremely small number of sequences (and possibly onlyone molecule of nucleic acid) corresponding to the highest affinitynucleic acids exist in the candidate mixture, it is generally desirableto set the partitioning criteria so that a significant amount of thenucleic acids in the candidate mixture (approximately 5-50%) areretained during partitioning.

4) Those nucleic acids selected during partitioning as having therelatively higher affinity for the target are then amplified to create anew candidate mixture that is enriched in nucleic acids having arelatively higher affinity for the target.

5) By repeating the partitioning and amplifying steps above, the newlyformed candidate mixture contains fewer and fewer unique sequences, andthe average degree of affinity of the nucleic acids to the target willgenerally increase. Taken to its extreme, the SELEX process will yield acandidate mixture containing one or a small number of unique nucleicacids representing those nucleic acids from the original candidatemixture having the highest affinity to the target molecule.

The SELEX process provides high affinity ligands to a target molecule.This represents a singular achievement that is unprecedented in thefield of nucleic acids research.

Certain chemical modifications of the nucleic acid ligand which can bemade to increase the in vivo stability of the nucleic acid ligand or toenhance or to mediate the delivery of the nucleic acid ligand. See,e.g., U.S. patent application Ser. No. 08/117,991, filed Sep. 9, 1993,entitled “High Affinity Nucleic Acid Ligands Containing ModifiedNucleotides,” abandoned in favor of U.S. patent application Ser. No.08/430,709, now U.S. Pat. No. 5,660,985, that describes oligonucleotidescontaining nucleotide derivatives chemically modified at the 5- and2′-positions of pyrimidines. Modifications of the nucleic acid ligandscontemplated in this invention include, but are not limited to, thosewhich provide other chemical groups that incorporate additional charge,polarizability, hydrophobicity, hydrogen bonding, electrostaticinteraction, and fluxionality to the nucleic acid ligand bases or to thenucleic acid ligand as a whole. Such modifications include, but are notlimited to, 2′-position sugar modifications, 5-position pyrimidinemodifications, 8-position purine modifications, modifications atexocyclic amines. substitution of 4-thiouridine, substitution of 5-bromoor 5-iodo-uracil; backbone modifications, phosphorothioate or alkylphosphate modifications, methylations, unusual base-pairing combinationssuch as the isobases isocytidine and isoguanidine and the like.Modifications can also include 3′ and 5′ modifications such as capping.

The modifications can be pre- or post-SELEX process modifications.Pre-SELEX process modifications yield nucleic acid ligands with bothspecificity for their SELEX target and improved in vivo stability.Post-SELEX process modifications made to 2′-OH nucleic acid ligands canresult in improved in vivo stability without adversely affecting thebinding capacity of the nucleic acid ligand.

Other modifications are known to one of ordinary skill in the art. Suchmodifications may be made post-SELEX process (modification of previouslyidentified unmodified ligands) or by incorporation into the SELEXprocess.

Ligand Beacons—Assay Design

In one embodiment of the invention, a ligand beacon is used to detect anucleic acid ligand that is or is not bound to its cognate target. Theligand beacon preferably consists of a single-stranded DNA molecule thatassumes a stem-loop structure in solution (FIG. 2A). In this embodiment,the stem of the ligand beacon is formed by the intramolecularbase-pairing of two antiparallel strands of the nucleic acid. The 5′terminus of one strand is linked to the 3′ terminus of the other strandwith a loop of single stranded DNA. These nucleic acid molecules can berapidly synthesized as single-stranded oligonucleotides with the generalstructure:

5′ AAA - - - A′A′A′ 3′

wherein sequence A′ is both complementary in sequence and reversed inorientation relative to A. When heat-denatured and slowly cooled, thisoligonucleotide will form a stem-loop structure wherein the dashed lineforms the loop, and wherein A and A′ pair to form the stem. The loopdomain comprises sequences that are at least partially complementary toa region of the nucleic acid ligand. In preferred embodiments, thesequences are chosen such that they can only hybridize to one anotherwhen the nucleic acid ligand is not bound to its cognate target.Furthermore, when the ligand beacon hybridizes to the nucleic acidligand, the nucleic acid ligand can no longer bind to its cognatetarget. In particularly preferred embodiments, the loop of the ligandbeacon binds to a sequence in the nucleic acid ligand that is at leastabout 20 nucleotides long; the stem region of the ligand beacon ispreferably shorter.

The formation of the intermolecular duplex between the loop of theligand beacon and the target-free nucleic acid ligand is energeticallyfavored because the resulting duplex is longer, and hence more stable,than the intramolecular duplex. As the loop sequence and the nucleicacid ligand form a duplex, torsional forces are developed in the ligandbeacon.These forces are transmitted to the stem region which unwinds inresponse, usually starting at the base of the stem where the termini arelocated. One base pair in the stem is unwound for each new base pairthat is made between the ligand beacon and the nucleic acid ligand.Thus, nucleotide positions that were adjacent to one another on oppositesides of the stem become separated. In particular, because unwindingbegins at the base of the stem, the termini of the ligand beacon becomewidely separated.

In some embodiments, nucleotide positions in the ligand beacon thatbecome separated from one another are labeled with an energy transferpair. The preferred energy transfer pair of the instant inventioncomprises a quenching group and a fluorescent group. In preferredembodiments, the nucleotide positions on the ligand beacon that arelabeled with the quenching group and the fluorescent group are chosenfrom those that form the intramolecular stem. In especially preferredembodiments, the 5′ and 3′ termini of the ligand beacon are labeled withthese groups, as the termini become widely separated upon hybridizationto the nucleic acid ligand.

The fluorescent group and the adjacent quenching group take part inenergy transfer. In some instances, the energy transfer occurs throughfluorescence resonance energy transfer (FRET). FRET takes place whenfluorescence emission from a fluorescent group is transferred to anadjacent group that somehow modifies the signal (in this case, quenchingthe signal). This effect is strongly dependent on the distance betweenthe two groups, such that when separated by a critical distance. FRETdoes not take place, and the fluorescence emission is unmodified. FREIalso requires that the emission spectrum of the fluorescent groupoverlaps with the absorbance spectrum of the modifying group.

The principle behind an assay that employs a molecular beacon and anucleic acid ligand based aptamer to detect molecular targets isschematically illustrated in FIGS. 2A-2C. With reference to FIG. 2, anucleic acid-based aptamer acts as a sensor that detects the presence ofa specific target molecule, for example, a protein, and in turn,communicates with the molecular beacon. Here, the nucleotide sequence inthe loop of the molecular beacon is complementary to a nucleotidestretch within an aptamer such that the hybridization of the two specieswill lead to the generation of a fluorescence signal (FIG. 2A). When theaptamer-specific molecular target is present in excess, the aptamerbinds to it with high affinity, making the aptamer unavailable forsubsequent hybridization with the molecular beacon (FIG. 2B). As aresult, the molecular beacon remains dark when the concentration of theaptamer-specific target exceeds the concentration of the aptamer. Whenthe target concentration is limiting, the amount of free aptameravailable for hybridization with the molecular beacon is inverselyproportional to the amount of the target present (FIG. 2C). Hence, theamount of fluorescence signal generated by a fixed amount of molecularbeacon and a fixed amount of aptamer is inversely proportional to theconcentration of the target.

The detection range of the assay is set by the concentration of theaptamer/ligand beacon pair. Higher concentrations of proteins aredetected by increasing the concentration of these two species. The lowerlimit of detection of the assay is dictated by the inherent sensitivityof the reporter, the fluorophore, attached to the ligand beacon.

In the instant invention, the preferred fluorescent groups arefluorescein, tetramethylrhodamine and5-[(2-aminoethyl)arnino]napthalene-1-sulfonic acid (EDANS). Thepreferred quencher is (4-dimethylaminophenylazo)benozoic acid (DABCYL).When DABCYL and fluorescein or EDANS are close enough together for FRETto occur, DABCYL absorbs light emitted from the fluorescein or EDANS,and dissipates the absorbed energy as heat. As mentioned above, thiseffect is strongly dependent on the distance between the group. Forexample, at separations greater than 60 Å, DABCYL is unable to quenchthe fluorescence from EDANS. DABCYI, itself is non-fluorescent at thewavelengths used to excite EDANS or fluorescein.

In other embodiments, the fluorescent group and the quenching group takeplace in a form of energy transfer termed direct energy transfer. Directenergy transfer occurs when the fluorescent group and the quenchinggroup directly perturb each others electronic structure. When a directtransfer takes place, it is possible for a quenching group to quench ata much higher efficiency and over a broader spectrum than in FRET.Indeed, it has been reported that paired-groups that do not even displayFRET, such as Texas Red and DABCYL, can be made to undergo direct energytransfer, leading to the efficient quenching of the fluorescence groupby the other group. For example, it has been reported that under suchcircumstances, the quenching group DABCYL can quench almost allfluorophores (with emission spectra ranging from 475 nm-615 nm) withclose to 100% efficiency (Tyagi et al. (1998) Nature Biotechnology16:49-53).

In one preferred embodiment, fluorescein and DABCYL function as a directenergy transfer pair when present at the 5′ and 3′ termini, even thoughthey are not an efficient FRET pair. In other embodiments, nucleotidepositions that form an individual base pair in the stem are labeled withthe fluorescent group and the quenching group. Labeling at thesepositions also allows direct energy transfer to take place. It is evenpossible to get fluorescence quenching when two identical fluorescentgroups, such as two fluorescein groups, are sufficiently close together.

There is no limitation in the present invention as to the nature of theenergy transfer pair, and there is no limitation as to the exactmechanism by which they function together. All that is required is thatthe spectral properties of the energy transfer pair change in somemeasurable way as the distance between the individual members of theenergy transfer pair is varied.

It is possible to label the ligand beacon with more than one of eachmember of an energy transfer pair. For example, in some embodiments, twoor more nucleotides are labeled with fluorescent groups and the samenumber of nucleotides are labeled with quenching groups. In preferredembodiments, all of the fluorescent groups are attached to thenucleotides that comprise one strand of the stem, and all of thequenching groups are attached to the nucleotides that comprises theother strand. In these embodiments, more than one base pair in the stemis labeled with both a fluorescent group and a quenching group. Suchligand beacons may give an increased signal relative to singly-labeledligand beacons upon unwinding of the stem.

Where more than one fluorescent group or more than one quenching groupis used, it is not required that there be an equal number of the twogroups. For example, the ligand beacon can be labeled with onefluorescent group and two quenching groups. If the sites of labeling aresufficiently close to one another, then more efficient quenching of thefluorescent group would be expected to result. Alternatively, if a givenquenching group is capable of quenching more than one fluorescent group,then the separation of a single effective quenching group from multiplefluorescent groups would be expected to give an increased signalrelative to separation from a single fluorescent group.

Labeling the ligand beacons with energy transfer pairs can beaccomplished easily by standard methods well known in the art. Forexample, it is possible to incorporate the fluorescent group fluoresceininto the ligand beacon at the 5′ end during automated oligonucleotidesynthesis of the sequence. The quenching group DABCYL can be attached tothe ligand beacon by first incorporating an amino group at the 3′ endduring oligonucleotide synthesis, and then reacting the amino groupafter synthesis with the succinimidyl ester of DABCYL in anhydrousN,N-dimethyl formamide. Alternatively, DABCYL can be incorporateddirectly into the ligand beacon during oligonucleotide synthesis. It isimportant to note that these methods can be adapted to place the membersof the energy transfer pair at any location desired in the ligandbeacon. In some embodiments it may not be useful to have the labels atthe termini. In some instances, for example, it may be preferable tolabel the stem of the ligand beacon at positions other than the 5′ and3′ termini. This is because under certain conditions, the termini of theligand beacon may temporarily unwind in the absence of free nucleic acidligand; which can lead to background fluorescence.

It is possible to use fluorescent groups with molecules other thanquenching groups. For example, a fluorescent group can be placed next toa modifying group that shifts the emission wavelength, polarizes theemission or even enhances it. All of these effects result from FRET.

Using the instant methods it is possible to simultaneously detectmultiple target molecules in a test solution using ligand beacons. Inthis method, each target molecule is recognized by a distinct nucleicacid ligand and each nucleic acid ligand can hybridize to a differentligand beacon. Each ligand beacon in the assay has at least a differentloop sequence, specific for a particular nucleic acid ligand. However,it is not necessary that each ligand beacon has a different stemsequence, as the stem sequence does not impart the specificity of theligand beacon, so it is possible to use a common stem for every ligandbeacon. In addition, each ligand beacon is labeled with at least adifferent fluorescent group. For example, to detect two differenttargets, two different nucleic acid ligands and two different ligandbeacons are required. For example, one ligand beacon may be labeled withfluorescein and DABCYL, and the second labeled with rhodamine andDABCYL. Therefore, the concentration of the two targets can bedetermined in the test solution by monitoring the increase in bothfluorescein and rhodamine emission.

It is important to note that it is not necessary to have any structuralinformation about a nucleic acid ligand when designing its cognateligand beacon. Given the rapidity with which one can synthesize theligand beacons, only simple, routine experimentation is required todesign several different ligand beacons for each nucleic acid ligand,each ligand beacon recognizing a sequence that it at least partiallyunique. The candidate ligand beacons can be quickly tested to determinewhich one has the desired activity.

As described above, preferred embodiments use ligand beacons that canbind to nucleic acid ligands only when the nucleic acid ligand is notbound to its target. However, the invention also includes ligand beaconsthat function in the converse manner. Specifically, the invention alsoincludes ligand beacons that can only hybridize to nucleic acid ligandsthat are bound to their cognate targets. For example, it is possible toobtain nucleic acid ligands that adopt a primary conformation in theabsence of target, but undergo a conformational change upon targetbinding. Such a conformational change may cause regions of the nucleicacid ligand that are initially double-stranded to becomesingle-stranded. The ligand beacon can hybridize to thesesingle-stranded regions, but not when they are double-stranded. As aresult, the increase in fluorescence intensity that occurs upon mixingthe nucleic acid ligand, the ligand beacon and the target is directlyproportional to the amount of the target.

In other embodiments, the ligand beacon has a structure in whichnucleotide positions that are initially separated become adjacent uponhybridizing to the nucleic acid ligand. If these nucleotide positionsare labeled as described above with a fluorescent group and a quenchinggroup, then hybridization to the nucleic acid ligand results in adecrease in the ligand beacon's fluorescence emission.

Although the preferred ligand beacons of the invention have a stem-looparchitecture, there is no limitation on the structure of ligand beacons.Any nucleic acid structure that undergoes a change in configuration uponhybridizing to a nucleic acid ligand wherein individual nucleotides moverelative to one another in a reproducible manner is contemplated herein.It is possible to stack more than one G-quartet on top of each otherunder appropriate ionic conditions. In this embodiment of the invention,the nucleotides that are located between the G-quartet residues comprisethe nucleic acid sequences complementary to the nucleic acid ligand. TheG-quartet residues are labeled with the energy transfer pair(s); uponhybridization of the ligand beacon to the nucleic acid ligand, theG-quartet is disrupted, and the energy transfer pair(s) are separated.

In order to determine the concentration of a target molecule in a testmixture, it is preferable to first obtain reference data in whichconstant amounts of ligand beacon and nucleic acid ligand are contactedwith varying amounts of target. The fluorescence emission of each of thereference mixtures is used to derive a graph or table in which targetconcentration is compared to fluorescence emission. For example, aligand beacon that a) hybridizes to a target-free nucleic acid ligand;and b) has a stem-loop architecture with the 5′ and 3′ termini being thesites of fluorescent group and quenching group labeling, could be usedto obtain such reference data. Such a ligand beacon would give acharacteristic emission profile in which the fluorescence emissiondecreases as the target concentration increases in the presence of aconstant amount of ligand beacon and nucleic acid ligand. Then, a testmixture with an unknown amount of target would be contacted with thesame amount of first nucleic acid ligand and second ligand beacon, andthe fluorescence emission would be determined. The value of thefluorescence emission would then be compared with the reference data toobtain the concentration of the target in the test mixture. In someembodiments, the nucleic acid ligand becomes covalently attached to itstarget molecule in the assay. Methods for obtaining nucleic acid ligandswith this capability are described in U.S. patent application Ser. No.08/123,935. filed Sep. 17, 1993. entitled “Photoselection of NucleicAcid Ligands,” now abandoned (see U.S. patent application Ser. No.08/612,895, filed Mar. 8, 1996. entitled “Systematic Evolution ofLigands by Exponential Enrichment: Photoselection of Nucleic AcidLigands and Solution SELEX, now U.S. Pat. No. 5,763,177), which arespecifically incorporated herein in their entirety.

The assays that are possible using ligand beacons are far simpler thanconventional techniques for detecting non-nucleic acid target molecules.The assays require only three manipulations: a) addition of the nucleicacid ligand(s); b) addition of the ligand beacons; and c) measurement ofthe fluorescence. In many embodiments, there is no need to perform anywashing steps to remove background signal, unlike the ELISA assays knownin the art. Therefore, the present invention provides a single commonmethod that can be applied to virtually any target molecule. Because ofthe simplicity of the assay, it is particularly well suited tohigh-throughout automated analysis for medical diagnostic purposes.

In some embodiments, the ligand beacons are used in assays in whichnucleic acid ligands are attached to the surface of a solid support.Methods for attaching nucleic acids to solid supports are well known inthe art. In these assays, the fluorescence emission from the solidsupport is monitored after the solid support is contacted with the testmixture suspected of containing the target, and the ligand beacon. It isalso possible to use multiple ligand beacons in assays in which aplurality of different nucleic acid ligands are attached to spatiallydiscrete addresses on a solid support forming an array. Nucleic acidligand arrays are described in U.S. patent application Ser. No.08/990,436, filed Dec. 15, 1997, entitled “Nucleic Acid LigandDiagnostic Biochip,” which is specifically incorporated herein byreference in its entirety. These assays require that each nucleic acidligand is recognized by a different ligand beacon with at least a uniqueloop sequence and a unique fluorescent group, as described above.Measuring the fluorescence emission profile of each address on the arrayreveals the concentration of each target molecule.

In still further embodiments, one or more ligand beacons are attached tothe solid support. Each ligand beacon can be attached via one of itstermini by a spacer molecule to allow the ligand beacon to adopt theappropriate conformations without hindrance from the underlying solidsupport. A test mixture is contacted with one or more nucleic acidligands and the mixture is contacted with the solid support. Again,measurement of the fluorescent emission profile at each address of thearray reveals the concentration of each target molecule in the testmixture.

The present invention also provides kits for the detection of particulartargets in test mixtures. The kit comprises separate containerscontaining solutions of a nucleic acid ligand to the particular target,and containing solutions of the appropriate ligand beacon. In someembodiments, the kit comprises a solid support to which is attached thenucleic acid ligand to the particular target. In further embodiments,the kit further comprises a container containing a standardized solutionof the target. With this solution, it is possible for the user of thekit to prepare a graph or table of fluorescence units vs. targetconcentration; this table or graph is then used to determine theconcentration of the target in the test mixture.

Cascade Hybridization

In one embodiment of the instant invention, a set of at least threesingle-stranded nucleic acids is synthesized, each comprising sequencesthat can form a stem-loop motif. The sequences of the antiparallel armsthat form the stem are chosen according to the following criteria: (i)each arm is only partially complementary to the opposing arm on the samemolecule with which it forms an intramolecular double helix (the stem);and (ii) each arm is perfectly complementary to one of the arms of atleast one other stem-loop nucleic acid. Any sequences with the abilityto form imperfect intramolecular base-pairs and perfect intermolecularbase-pairs are contemplated. The sequences are represented schematicallyin FIG. 10 as A, B and C and their respective complements A′, B′ and C′.

FIG. 10 shows that A pairs imperfectly with B, B′ pairs imperfectly withC and C′ pairs imperfectly with A′. The loop region of each nucleic acidis represented as a curve. In one embodiment, the loop of stem-loop Icomprises probe sequences that are complementary to a target nucleicacid that one would like to detect. Upon hybridization of the probesequence to its cognate target, the arms AB will become unpaired. Thiswill occur because the base pair formation between the loop and thetarget sequence is energetically more favored than that between the armsof the stem, and the structure adopted by the loop in order to bind tothe target is not compatible with the stem structure. The sequences Aand B of stem-loop I in FIG. 10 will now be available to hybridize withtheir complementary sequences. In FIG. 10, B can pair with the sequencesB′ from stem-loop II, which in turn makes the sequence C available forhybridization with the C′ sequence from stem-loop III. In each case, theformation of the perfect intermolecular double helices is energeticallyfavored once the base pairs between A and B are disrupted.

This cascade of hybridization will propagate in both directions (FIG.11) until one of the nucleic acids becomes depleted from thehybridization mixture. It can therefore be seen that a singlehybridization event between the loop sequence of the probe nucleic acidand the target sequence sets off a chain of hybridization reactionsbetween the stem-loop nucleic acids. The formation of the multimolecularcomplex depends only on the presence of a single copy of the targetmolecule. Therefore, the cascade hybridization technique provides anexquisitely sensitive method for detecting the presence of a targetmolecule.

In another embodiment, the nucleic acid that initiates the hybridizationcascade comprises a nucleic acid ligand. This ligand or a nucleic acidattached thereto will undergo a conformational change upon binding to atarget molecule, which change allows the chain of hybridization tobegin. In a preferred embodiment, the nucleic acid ligand will becontained within the loop sequence of a stem-loop nucleic acid. Asdescribed above, the initiating event is the energetically-favoredbinding of the loop sequences to the target molecule, which forces thenucleic acid to adopt a conformation wherein the stem is dissociated.Stem-loop nucleic acids that undergo stem dissociation upon binding to atarget molecule are described in U.S. patent application Ser. No.08/134,028, filed Oct. 7, 1993, entitled “High-Affinity Nucleic AcidLigands that Discriminate Between Theophylline and Caffeine,” nowabandoned (see U.S. Pat. No. 5,580,737).

In a related embodiment, the set of nucleic acids will further comprisea nucleic acid with the same sequence as the initial probe nucleic acid,but without the target molecule-binding site. This nucleic acid willtherefore not participate in the initial target binding reaction, butwill participate in the cascade hybridization. Use of this nucleic acidat a higher concentration than the target-binding nucleic acid willprevent equilibrium exchange of the target molecule between anestablished multimolecular cascade hybridization product and a free,unhybridized target-binding nucleic acid. This embodiment will preventthe loss of established cascade products from the surface of solidsupports (see below).

In any of the preceding embodiments, more than three different nucleicacid sequences may be used, as long as the sequences thereof willpromote cascade hybridization.

Although preferred embodiments use intramolecular double-helices andstem-loop nucleic acids, cascade hybridization will also be possiblewith other nucleic acid structures. All that is required is that thenucleic acids undergo a conformational change upon binding so thatsequences previously unavailable for hybridization become available. Anynucleic acid that contains a binding site for a target molecule andundergoes a conformational change when it binds to said target moleculeis suitable for use as the cascade-initiating nucleic acid. Any nucleicacids that can bind to said target-bound cascade-initiating nucleic acidand then propagate the cascade as described above are suitable for useas the hybridizing nucleic acids.

Signal Amplification Through Cascade Hybridization

In one embodiment, each nucleic acid described above is labeled with anenergy transfer pair. The two members of the transfer pair are attachedto nucleotides at location on the nucleic acid that are spatiallyadjacent only when the nucleic acid is not participating in a bindingreaction. Therefore, binding of the nucleic acid to either the targetmolecule or to the other nucleic acids results in the spatial separationof the energy transfer pair. The disruption of energy transfer can bedetected by spectroscopic techniques. In the case of the nucleic acidsdescribed above with a stem-loop motif, suitable positions for theenergy transfer pair members include, but are not limited to, the 5′ andthe 3′ termini of each nucleic acid.

In a preferred embodiment, the energy transfer pair comprises aligand-beacon. In the unbound conformation, the signal from thefluorescent group is quenched. When the nucleic acid participates in abinding reaction, the fluorescence signal is no longer quenched, and canbe detected by any of the means known in the art. Termination ofquenching will occur at the initial target molecule binding event, andwill also occur at each consequent intermolecular helix formation event.Therefore, the binding of a single nucleic acid to a single targetmolecule will result in the generation of a fluorescent signal thatincreases in magnitude as the cascade progresses (FIG. 12). Any energytransfer pair known in the art is suitable for incorporation into thenucleic acids in this embodiment. as discussed above.

One advantage of the energy transfer approach is that only cascadehybridization reactions will lead to the production of aspectroscopically-detectable signal. For example, if the nucleic acidsare labeled with a fluorescent group and a quenching group, and ifhybridization occurs between the individual nucleic acids in the absenceof target molecule-binding, then the individual quenching groups will bespatially adjacent to the fluorescent groups on the hybridizing nucleicacids (FIG. 13) and fluorescent signal will be generated.

In other embodiments, the nucleic acids are labeled with animmunologically detectable probe, such as digoxigenin. The presence ofdigoxigenin can be detected using fluorescently-labeled antibodies thatbind specifically thereto, or by using a sandwich assay.

Use of Cascade Hybridization

In one embodiment, the cascade hybridization system is used to detectthe presence of a target molecule in a test solution. A first nucleicacid with a binding site for the target molecule is added to the testsolution together with the cascade hybridization nucleic acids describedabove. If the target molecule is present, then a cascade hybridizationcomplex will form. In one embodiment, this nucleic acid complex will bedetected by the standard gel electrophoresis methods known in the art.In another embodiment, the nucleic acids will be labeled with afluorescent group and a quenching group as described above. The presenceof the complex, and hence of the target molecule, can be detected bycomparing the fluorescence of the solution with that of a solutioncontaining only the set of nucleic acids.

In another embodiment, the target-binding nucleic acid, containing thebinding site for the target molecule, is immobilized on the surface of asolid support, such as a biochip. The nucleic acid is attached at eitherthe 3′ or the 5′ end to functional groups displayed on the surface ofthe biochip. Methods for attaching nucleic acids to biochips are wellknown in the art, and include methods for attaching different nucleicacids to discrete locations on the same biochip. The biochip is thencontacted with a test mixture suspected of containing target moleculesto which the first nucleic acid can bind. Following removal of unboundmaterial from the biochip by washing, the biochip will be contacted witha solution containing the three sets of nucleic acids labeled asdescribed above with an energy transfer pair. If the biochip-boundnucleic acid binds to target molecule, then a multimolecular complexwill form on the biochip. The presence of this complex can be detectedby measuring fluorescence on the biochip. Such a biochip can be used todetect exceedingly rare molecules in test mixtures, and will haveutility in diagnostic and prognostic medical screening, and inenvironmental testing.

In a related embodiment, a plurality of different species oftarget-binding nucleic acids will be localized to discrete locations onthe biochip. Each species of target-binding nucleic acid has a uniqueloop region sequence with a specific affinity for a different targetmolecule, and a common stem sequence. The biochip is then contacted witha test mixture suspected of containing one or more of the targetmolecules to which the target-binding nucleic acids can bind. Followingincubation with the nucleic acid set, the fluorescence of each locationon the biochip is measured. In this way, the presence of multiple targetmolecules can be determined simultaneously. This method will be usefulin medical screening applications, where the diagnosis or prognosis of aparticular disease requires the measurement of several differentmolecules contained in the patient's blood or urine.

Use of Cascade Hybridization in situ

In one embodiment, cascade hybridization is used in situ to give spatiallocalization data for a target molecule. For example, the location of anRNA molecule in a fixed tissue sample can be determined if thetarget-binding nucleic acid comprises sequences complementary to thisRNA molecule. The use of the fluorescence quenching cascade system inthis scenario would result in the formation of a highly fluorescentsignal at the site of deposition of the RNA molecule of interest. Byusing a nucleic acid ligand as the target-binding nucleic acid, thepresence of virtually any target molecule can be determined in suchfixed specimens. These techniques are vastly more sensitive than thesandwich immunoassays and in situ hybridization techniques known in theart, as the cascade signal amplification system can detect the presenceof even a single copy of the target molecule.

Antibody-linked Cascade Hybridization

In a related embodiment, the cascade hybridization system is used todetect the binding of an antibody to a specific target molecule. Theantibody is conjugated to an oligonucleotide, and the antibody then usedto probe the sample suspected of containing the target molecule ofinterest. A set of cascade hybridization nucleic acids is then used todetect the presence of the oligonucleotide, and hence the targetmolecule. The first cascade nucleic acid will hybridize to theantibody-bound oligonucleotide. This cascade nucleic acid will then beavailable for hybridization with other members of the set of cascadenucleic acids and cascade hybridization will ensue as described above.If the cascade nucleic acids are fluorescently labeled, or are labeledwith both a quenching group and a fluorescent group, then a fluorescentsignal will be produced.

This embodiment can be used in situ to give fluorescent localizationdata for extremely scarce target molecules that would not be detectedusing the standard sandwich immunoassays known in the art. Inparticular, the cascade system can be used to detect the binding of evena signal antibody to its target molecule; this sensitivity is notpossible using standard immunoassays. This embodiment can also be usedin in vitro immunoassays as a replacement for enzyme-linkedimmunoassays. Alternatively, the nucleic acids can be conjugated to anelectron-dense label, such as a gold microparticle, and used inimmunoelectron microscopy.

Example 1 describes the synthesis of a ligand beacon to human plateletderived growth factor (PDGF) (see FIG. 3B, SEQ ID NO:2).

Example 2 describes the synthesis of a ligand beacon to thermophilusaquaticus (Taq) DNA polymerase (see FIG. 3B, SEQ ID NO:4).

Example 3 illustrates the use of ligand beacon assays. Two differentpairs of aptamers and aptamer-specific ligand beacons were analyzed inthis example. A DNA aptamer that binds to the AB-dimer of humanplatelet-derived growth factor (PDGF) with an equilibrium dissociationconstant (K_(d)) of 0.15 nM (Green et al. (1996) Biochemistry35:14413-14424) was used for the first experiment described in Example3. This aptamer has the capacity to fold into a three-way helix junctionwith a three-nucleotide loop at the branch point (FIG. 3A, SEQ IDNO:10). Another DNA aptamer that recognizes DNA polymerase from Thermusaquaticus (Taq) polymerase (Dang and Jayasena (1996) J. Mol. Biol.264:268-278), was used for the second experiment described in Example 3.This aptamer has the potential to fold into a stem-loop structurecontaining several bulges in the stem (FIG. 3A, SEQ ID NO:9). Theresults of these assays are set forth in FIGS. 4A and B (PDGF and Taq,respectively), which depicts the fluorescence signal produced by a fixedconcentration of ligand beacon in the presence of increasingconcentration of an aptamer sequence having a predicted secondarystructure. In both cases, the fluorescence signal produced by a fixedamount of ligand beacon increased as the concentration of theappropriate aptamer was increased in the reaction mixture (FIGS. 4A andB; closed circles), indicating that the ligand beacons can haveproductive interactions with aptamers containing complementarynucleotide regions. In these experiments short single stranded DNAsequences complementary to the loops of the two ligand beacons (FIG. 3B:sequence regions shown in bold) were used as controls. The shortsequence complementary to the Taq ligand beacon is single-stranded,whereas the sequence complementary to the PDGF ligand beacon may containa short base paired region. Both of these short sequences are devoid ofextra nucleotides present in aptamers that do not participate inhybridization with ligand beacons. The signal generated by the aptamers(FIG. 4; closed circles) is slightly lower than that produced with thesame concentration of the short single-stranded target sequences (FIG.4; open circles). This result suggests that extra nucleotide regions inaptamers that do notparticipate in hybridization have very littleinterference in intermolecular association.

When wrong combinations of aptamer/ligand beacon pairs were mixed, nofluorescence signal above the background level was generated (FIGS. 4Aand B; stars), indicating the specificity of aptamer ligand beaconinteractions. Moreover, these experiments were carried out in thepresence of vast excess of tRNA, further indicating the lack ofinterference on signal generation by nonspecific nucleic acids. Theseresults, reflecting the extreme specificity of molecular beacons, areconsistent with observations made by others (Kostrikis et al. (1998)Science 279:1228-1229; Piatek et al. (1998) Nature Biotechnol.16:359-363; Tyagi et al. (1998) Nature Biotechnol. 16:49-53; Tyagi andKramer (1996) Nature Biotecnol. 14:303-308).

The Taq ligand beacon targets a 24 nucleotide stretch of which 22nucleotides constitute the single stranded loop region in the middle ofthe Taq aptamer. The hybridization of this pair will result in acontinuous duplex containing 24 base pairs, the formation of which mightmelt the imperfect helix containing 11 base pairs in the aptamer (FIG.3A). In the case of the PDGF aptamer, hybridization with its ligandbeacon is expected to form a duplex with 24 contiguous base pairs at theexpense of 9 base pairs found in the aptamer (FIG. 3A). The formation ofenergetically favored long helical regions with contiguous base pairs isthe driving force for the intermolecular hybridization between aptamersand ligand beacons. The results shown in FIG. 4 were obtained uponincubating the aptamer/ligand beacon pairs at 37° C. When incubated atroom temperature the amount of fluorescence signal was significantlylower than that observed at 37° C. (data not shown). This suggests thatthe intermolecular hybridization between the aptamer and its ligandbeacon is favored at higher temperature, conditions that enhance frayingor breathing of structured molecules. Overall, these results indicatedthat an aptamer/ligand beacon pair can be used to develop an assay asillustrated in FIG. 2.

Example 4 describes the synthesis of ligand beacons to human L-Selectin(SEQ ID NO:6) and human P-selectin (SEQ ID NO:8). (see FIG. 3B).

Example 5 (FIGS. 5-8) illustrates protein detection using ligand beaconassays in buffer. FIG. 5 depicts the results using PDGF aptamer (SEQ IDNO:10) and its ligand beacon (SEQ ID NO:2) to detect PDGF AB-dimer. Ascan be seen in FIG. 5A, a decrease in fluorescence signal produced bythe same concentration of the ligand beacon was observed with increasingconcentration of the PDGF AB-dimer. This result is consistent with thescheme illustrated in FIG. 2. As the concentration of the PDGF proteinis increased, increasing amounts of aptamer molecules are sequestered bythe protein due to high affinity interaction. As a result, less and lessaptamer molecules become available to generate the fluorescence signalupon hybridization with the ligand beacon. In the assay illustrated inFIG. 5A, the PDGF aptamer was first incubated with the PDGF AB-dimerbefore the ligand beacon was added. No noticeable change in signal wasobserved 10 minutes or 4 hours after adding the ligand beacon (data notshown), indicating that the excess free ligand beacon present in thereaction did not significantly disturb the equilibrium of aptamer targetbinding.

When the ligand beacon was added to the aptamer before the protein wasadded no decrease in the fluorescence signal was observed (FIG. 5B,(open circles)). In this case, as expected, the hybridization of theligand beacon to the aptamer generated a high fluorescence signal. Thisassociation between the aptamer and the ligand beacon destroyed theability of the aptamer to bind to the target. Addition of the targetprotein to the ligand beacon alone did not change the signal intensityeither (FIG. 5B, (closed circles)). Results of these control experimentsindicate the following: 1) the decrease in fluorescence signal requiresthe presence of all three components added in the right order: 2) thedecrease in fluorescence signal observed in FIG. 5A is not due to thenonspecific interaction of the fluorophore with the protein that mighthave caused the quenching of fluorescence; 3) the native or thefunctional conformation of an aptamer must be preserved at the time ofits interaction with its target; and 4) as was expected, the stabilityof the ligand beacon aptamer hybrid is such that the addition of thetarget protein was unable to dissociate the aptamer from the hybrid.

Similar to the results obtained with the PDGF protein and itsaptamer/ligand beacon pair, a decrease in fluorescence signal with theincreasing concentration of Taq DNA polymerase added to itsaptamer/ligand beacon pair was also observed (FIG. 6). The results ofthese experiments are consistent with the proposed scheme for the ligandbeacon assay.

The assay concept was further tested with two different, but closelyrelated proteins; human L-selectin and human P-selectin. High affinityaptamers to these two human selectins have been recently described(Jenison et al. (1998) Antisense & Nucleic Acids Drug Dev. 8:265-279;O'Connell et al. (1996) Proc. Natl. Acad. Sci. USA 93:5883-5887). TheDNA aptamer that binds to L-selectin with a K_(d) of 1.8 nM has thepotential to fold into a stem-loop structure with an internal bulge(FIG. 3A, SEQ ID NO:11). For P-selectin, an RNA aptamer was chosen totest the feasibility of an RNA aptamer in the assay. The P-selectin RNAaptamer containing 2′-F pyrimidines has the potential to form astem-loop structure with several internal bulges (FIG. 3A, SEQ IDNO:12). The interaction of this particular aptamer with P-selectin has aK_(d) of 40 pM. Ligand beacons specific to both aptamers were designed(FIG. 3B, SEQ ID NOS:6 and 8, respectively).

Analogous to PDGF and Taq aptamer/ligand beacon pairs, the L-selectinaptamer/ligand beacon pair also showed productive hybridization at 37°C. producing a fluorescence signal (data not shown), indicating thefeasibility of using this pair in the assay. Additionally, as in thecase of the PDGF and Taq proteins, the fluorescence signal measured wasinversely proportional to the amount of L-selectin added (FIG. 7A). Inthe L-selectin assay, as well as in the PDGF assay, the fluorescencesignal approaches zero when the target concentration approaches theconcentration of the aptamer, suggesting that the concentration of theaptamer must be increased to detect high concentrations of the target.By increasing the aptamer and the ligand beacon concentration. it ispossible to detect high concentrations of the target (FIG. 7B). Hence,the range of the assay is dictated by the concentration of theaptamer/ligand beacon pair.

Example 5 also illustrates the use of a 2′-F-pyrimidine-containing RNAaptamer specific for human P-selectin in a ligand beacon assay. Similarto DNA aptamers. the 2′-F-pyrimidine containing RNA aptamer was able tohybridize efficiently to its ligand beacon at 37° C. to generatefluorescence signal in a concentration dependant manner (FIG. 8A).Furthermore, it was also possible to detect human P-selectin using thisaptamer/ligand beacon pair (FIG. 8B). The results of the assay based onan RNA aptamer to detect the P-selectin protein follows the same trendas the assays based on DNA aptamers, indicating that both DNA and RNAaptamers are equally suited for the ligand beacon assay. The specificityof the assay was also tested by exchanging the two closely relatedtarget proteins, L-selectin and P-selectin. As shown in FIG. 8C, therewas no significant decrease in the fluorescence signal when anincreasing concentration of the wrong selectin was added to a matchedpair of aptamer/ligand beacon; for example, P-selectin protein withL-selectin aptamer and L-selectin ligand beacon. These results indicatethat the assay specifically detects the correct target in the medium.Although specific interactions between all three components, the target,the aptamer and the ligand beacon are crucial, by and large, the overallspecificity of the assay rests primarily upon the aptamer-targetinteraction. Nonspecific sequestering of the aptamer could also lead tothe decrease in fluorescence signal. The lack of cross reactivity in thetwo selectin assays is primarily due to the specificity of aptamers thatwere selected to recognize the two selectins.

L-selectin and P-selectin, together with E-selectin, constitute a familyof homologous cell adhesion molecules called selectins. Although thesethree selectins are highly homologous, aptamers that have been selectedto bind one type do not bind the other two types with high affinity. TheL-selectin DNA aptamer used in this assay discriminates its binding toP-selectin and E-selectin by 9000-fold and 300-fold, respectively(O'Connell et al. (1996) Proc. Natl. Acad. Sci. USA 93:5883-5887). TheP-selectin RNA aptamer used in this study exhibits 2000-fold reducedaffinity to L-selectin (Jenison et al. (1998) Antisense & Nucleic AcidsDrug Dev. 8: 265-279). This high level of discrimination in targetbinding by the aptamers translated to the observed specificity in theligand beacon assay.

Example 6 illustrates the feasibility of using a ligand beacon assay tomeasure protein targets in plasma. Biological fluids are complexmixtures made up of constituents that generally tend to interfere indiagnostic assays. To avoid such interference, certain diagnostic assayformats, such as enzyme-linked immunosorbent assays (ELISA), include astep that captures (or separates) the analyte of interest frombiological fluids before being detected. Homogeneous assays, however,are able to detect analytes even in complex milieu of biological fluids,without a need for separation of analytes. Hence, homogeneous assays aresimple to use, attractive for high throughput applications and lessexpensive. Example 6 demonstrates that the ligand beacon assay allowshomogeneous detection of proteins directly in plasma, making the assaymore attractive for clinical diagnostic applications.

In Example 6, the target protein was added to human plasma containingthe aptamer mixed with tRNA. After incubating the reaction mixture for10 minutes at 37° C., the corresponding ligand beacon was added and theincubation was continued for another 10 minutes before fluorescence wasmeasured. As shown in FIGS. 9A-C, all three protein targets could bemeasured in human plasma. In each case, the decrease in fluorescencesignal observed in plasma in response to the increase in target proteinconcentration is analogous to what was observed in the buffer. Plasmacontains a variety of proteins and other metabolites at varyingconcentrations. Most importantly, these components did not appear tointerfere with the overall performance of the assay. Analogous to theassay carried out in the buffer, the detection range of the assay inplasma was also dictated by the concentration of the aptamer/ligandbeacon pair. The use of two different concentrations of aptamer/ligandbeacon pairs enabled detection of two different concentrations of targetproteins (data not shown). The ability to detect protein targets inplasma using the ligand beacon assay would be expected to expand itsutility.

Example 7 illustrates cascade hybridization.

The following examples are presented for illustrative purposes only andare not intended to limit the scope of the invention.

EXAMPLES

Methods and Materials

DNA oligonucleotide sequences were synthesized by standard solid phasechemical synthesis using cyanoethyl phosphoramidites and purified onpolyacrylamide gels run under denaturing conditions. The 2′-F-pyrimidinecontaining RNA aptamer was produced by run-off in vitro transcriptionmethod using T7 RNA polymerase and a PCR-derived DNA template (Milliganet al. (1987) Nucleic Acids Res. 15:8783-8798) as described (Davis etal. (1998) Nucleic Acids Research 26:3915-3924). The AB-dimer of thehuman platelet-derived growth factor (PDGF-AB), human L- and P-slectinwere purchased from R & D Systems. Taq DNA polymerase was obtained fromRoche Molecular Systems.

Ligand beacon sequences containing fluorescein at the 5′ end weresynthesized using fluorescein phosphoramidite (Glen Research). The solidphase synthesis of ligand beacons was initiated with 3′-amino-modifierC7 CPG (Glen Research) that incorporates reactive amine functionalgroups at 3′ ends to facilitate post-synthesis attachment of thequencher, 4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL) (Tyagi andKramer (1996) Nature Biotecnol. 14:303-308). After deprotection, theligand beacons were equilibrated in 300 μL of 0.3 M sodium acetate at3-5 mg/mL and incubated for 30-60 minutes at room temperature, thenprecipitated with ethanol. The DNA pellet was then dissolved in 100 μLof 100 mM sodium borate buffer (pH 9.3) and mixed with 10 mg ofsuccinimidyl ester of DABCYL (Molecular Probes) in 100 μL of anhydrousN,N-dimethyl formamide (DMF). The DABCYL conjugation reaction wasallowed to proceed for 30 minutes at room temperature. The reactionmixture was then passed through Centrex U-0.5 10K MWCO CentrifugalUltrafiltration Unit (Schleicher and Schuell) and the DNA retained onthe filter was thoroughly washed with DMF. DNA-DABCYL conjugate wasrecovered from the filter and purified on 10% polyacrylamide gels ranunder denaturing conditions. Ligand beacons (10-50 μM) in theappropriate assay buffer were heated to 90° C. and allowed to cool toroom temperature before use.

Alternatively, after deprotection, the oligonucleotide was equilibrated100 mM sodium borate buffer (pH 9.3) at 4 mg/mL and mixed with an equalvolume of the succinimidyl ester of (4-dimethylaminophenylazo)benozoicacid (DABCYL) in anhydrous DMF (5 mg/100 μL). The reaction was allowedto proceed for 30 minutes at room temperature. Unreacted DABCYL wasremoved from the derivatized oligonucicotide by passing the reactionmixture through a 5000 MW cutoff Centricon filter. Subsequently,derivatized oligonucleotide was purified by gel electrophoresis underdenaturing conditions. The ligand beacon was heated to 80° C. in PBSMbuffer (10.1 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 137 mM NaCl, 2.7 mM KCl, 1 mMMgCl₂, (pH 7.4)) and slowly cooled to room temperature before use.

Aptamer-Ligand Beacon Interaction

A fixed concentration (200 nM) of a ligand beacon was mixed withincreasing concentration of the corresponding aptamer in 100 μL volumeof the binding buffer containing 4 μM tRNA in a microtiter plate andincubated at 37° C. for 10 minutes before fluorescence signal wasmeasured. Fluorescence was measured at 530 nm after exciting at 488 nmmonochromatic laser light in 96-well format Vistra Fluorimager SIinstrument.

Ligand Beacon Assay

Aptamers at 10 μM concentration in the appropriate binding buffer wereheated to 90° C. and cooled to room temperature to facilitate secondarystructure formation. Serially diluted target protein (50 μL) either inthe appropriate buffer or in plasma were added to microtiter plate wellsin duplicate. Then, a fixed concentration of an aptamer mixed with tRNAat a concentration of four-fold higher than that of the aptamer in 25 μLvolume was added to each well. After 10 minutes incubation at 37° C., 25μL of the corresponding ligand beacon was added to the finalconcentration equivalent to that of the aptamer and the incubation wascontinued for another 10 minutes. Fluorescence in each well was measuredat 530 nm after exciting at 488 nm using monochromatic laser light in a96-well format Vistra Fluorimager SI.

The fluorescence signal intensity of each well was subtracted from thatof a background well that contained only the ligand beacon in theappropriate buffer or plasma. The background-subtracted signal wasplotted against the log concentration of the target protein. Nonlinearleast squares method was used for curve fitting with Kaleidagraph(Synergy Software. Reading, Pa.) using equation 5 as derived below.$\begin{matrix}\begin{matrix}\begin{matrix}\begin{matrix}\begin{matrix}{{P + A}\overset{\quad}{\rightleftharpoons}{P:A}} \\{K_{a} = \frac{\left\lbrack {P:A} \right\rbrack}{\lbrack P\rbrack \lbrack A\rbrack}}\end{matrix} \\{\left\lbrack P_{T} \right\rbrack = {\lbrack P\rbrack + \left\lbrack {P:A} \right\rbrack}}\end{matrix} \\{\left\lbrack A_{T} \right\rbrack = {\lbrack A\rbrack + \left\lbrack {P:A} \right\rbrack}}\end{matrix} \\{\left\lbrack {P:A} \right\rbrack = {{K_{a}\lbrack P\rbrack}\lbrack A\rbrack}}\end{matrix} & \lbrack 1\rbrack\end{matrix}$

Where, K_(a) is the equilibrium association constant for the interactionbetween the target protein P and the aptamer A. P_(T) represents thetotal protein concentration and A_(T) indicates the total aptamerconcentration. Equation 1 can be rearranged to obtain Equation 2.

[P:A]=K _(a)([P _(T) ]−[P:A])([A _(T) ]−[P:A])

[P:A]=K _(a)([P _(T) ][A _(T) ]−[P:A]([A _(T) ]+[P _(T)])+[P:A] ²)

[P:A] ² −[P:A]([A _(T) ]+[P _(T) ]+K _(a) ⁻¹)+([P _(T) ][A _(T)])=0  [2]

Equation 2 has the general form:

x ² −bx+c=0

Hence, $\begin{matrix}{\left\lbrack {P:A} \right\rbrack = {\frac{\left( {\left\lbrack A_{T} \right\rbrack + \left\lbrack P_{T} \right\rbrack + K_{d}} \right)}{2} - \sqrt{\frac{\left( {\left\lbrack A_{T} \right\rbrack + \left\lbrack P_{T} \right\rbrack + K_{d}} \right)^{2}}{4} - {\left\lbrack P_{T} \right\rbrack \left\lbrack A_{T} \right\rbrack}}}} & \lbrack 3\rbrack\end{matrix}$

Where,

K _(a)=1/K _(d)

Free aptamer interacts with the ligand beacon B, to generate the complexAB* that produces the fluorescence signal.

A+B→A:B*

*=Fluorescence signal

[A:B*]=[A]=[A _(T) ]−[P:A]  [4]

Using Equation 3, $\begin{matrix}{{Signal} = {\left\lbrack A_{T} \right\rbrack - \frac{\left( {\left\lbrack A_{T} \right\rbrack + \left\lbrack P_{T} \right\rbrack + K_{d}} \right)}{2} - \sqrt{\left. {\frac{\left. {\left( {\left\lbrack A_{T} \right\rbrack + P_{T}} \right\rbrack + K_{d}} \right)^{2}}{4} - {\left\lbrack P_{T} \right\rbrack \left\lbrack A_{T} \right\rbrack}} \right\rbrack}}} & \lbrack 5\rbrack\end{matrix}$

Example 1

Ligand Beacon for use with PDGF Nucleic Acid Ligand

A nucleic acid ligand to human platelet derived growth factor (PDGF)with the following sequence was obtained using the SELEX process asdescribed above:

5′-tgggagggcgcgttcttcgtggttacttttagtcccgt-3′ (SEQ ID NO:1)

The sequence in bold was used to design a ligand beacon with thefollowing sequence:

5′-F-gcgagaaagtaaccacgaagaagaacgcgcccctcgc-Q-3′ (SEQ ID NO:2)

wherein the bold sequence in the ligand beacon is complementary to thebold sequence in the nucleic acid ligand, and the underlined sequencesform the stem. The ligand beacon was labeled with fluorescein (F) at the5′ terminus and DABCYL (Q) at the 3′ terminus as described above.

Example 2

Ligand Beacon for use with Nucleic Acid Ligand to TAQ Polymerase

A nucleic acid ligand to Thermophilus aquaticus (TAQ) DNA Polymerasewith the following sequence was obtained through the SELEX methodologyas described above:

5′-tggcggagcgatcatctcagagcattcttagcgttttgttcttgtgtatga-3′ (SEQ ID NO:3)

The sequence in bold above was used to design a ligand beacon with thefollowing sequence:

5′-F-gcgagcaagaacaaaacgctaagaatgctctcgc-Q-3′ (SEQ ID NO:4)

wherein the bold sequence in the ligand beacon is complementary to thebold sequence in the nucleic acid ligand, and the underlined sequencesform the stem. The ligand beacon was labeled with fluorescein at the 5′terminus and DABCYL at the 3′ terminus as described above.

Example 3

Specificity of Ligand Beacon Interaction with Nucleic Acid Ligand

In order to test the specificity of the interactions of the ligandbeacons with their cognate nucleic acid ligands, the TAQ ligand beaconand the PDGF ligand beacon were contacted with either: their cognatenucleic acid ligand, a twenty nucleotides-long linear templateoligonucleotide sequence that is complementary to the ligand beacon, ora non-cognate nucleic acid ligand. The results are shown in FIGS. 4A andB. In the example shown in FIG. 4A. 200 nM PDGF-ligand beacon was mixedwith increasing concentration of PDGF nucleic acid ligand (closedcircles). 20-nt linear PDGF template (open circle) or TAQ nucleic acidligand (asterisks) in PBSM buffer consisting of 10.1 mM Na₂HPO₄, 1.8 mMKH₂PO₄, 137 mM NaCl₂, 2.7 mM KCl, 1 mM MgCl₂, 4 μM tRNA (pH 7.4) andincubated at 37° C. for 10 minutes before fluorescence was measured.Fluorescence was measured at 530 nm after exiting at 488 nm usingmonochromatic laser light in 96-well format Vistra fluorimager SI. Eachexperiment was done in duplicate.

In the example shown in FIG. 4B, 200 nM TAQ-ligand beacon was mixed withincreasing concentration of TAQ nucleic acid ligand (closed circles),20-nt linear TAQ template (open circle) and PDGF nucleic acid ligand(asterisks) in PBSM buffer containing 4 μM tRNA and incubated at 37° C.for 10 minutes before fluorescence was measured. Each experiment wasdone in duplicate.

In the example shown in FIG. 4C, TAQ-nucleic acid ligand (100 nM) wasmixed with increasing concentrations of PDGF in PBSM buffer containing 4KM tRNA at 37° C. for 10 minutes. Then 110 nM TAQ-ligand beacon wasadded, incubated for an additional 10 minutes at the same temperatureand fluorescence was measured. As illustrated in FIG. 4C, noconcentration-dependent signal reduction is observed when the wrongtarget protein is added. These results illustrate that signal generationis specific for the target protein.

Example 4

Ligand Beacons and Nucleic Acid Ligands to Selectins

Ligand beacons were synthesized for P-selectin and L-selectin nucleicacid ligands. The sequences of the appropriate nucleic acid ligands andtheir cognate ligand beacons are given below:

L-Selectin Nucleic Acid Ligand

5′-tagccaaggtaaccagtacaaggtgctaaacgtaatggcttcggcttac-3′ (SEQ ID NO:5)

L-Selectin Ligand Beacon

5′-F-gcgagtgtactggttaccttggctactcgc-Q-3′ (SEQ ID NO:6)

P-Selectin Nucleic Acid Ligand

5′-cucaacgagccaggaacaucgaggucagcaaacgcgag-3′ (SEQ ID NO:7)

P-Selectin Ligand Beacon

5′-F-gcgagctcgcgtttgctgacgtcgactcgc-Q-3′ (SEQ ID NO:8)

wherein the L-Selectin nucleic acid ligand is a 49-mer single-strandedDNA, and the P-Selectin nucleic acid ligand is a 38-mer RNA moleculecontaining 2′-F-substituted pyrimidines. The F represents fluoresceinand Q represents DABCYL. As in the previous examples, the ligand beaconswere synthesized with fluorescein at the 5′ end, and a free amino groupat the 3′ end. The free amino group was reacted with the succinimidylester of DABCYL in order to position DABCYL at the 3′ end of the ligandbeacon.

Example 5

Protein Detection using Ligand Beacon Assays

PDGF AB-Dimer

The PDGF ligand beacon was used in an assay in which 160 nM PDGF nucleicacid ligand (SEQ ID NO:1) was mixed with an increasing concentration ofPDGF AB-dimer in PBSM buffer [10.1 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 137 mMNaCl, 2.7 mM KCl, 1 mM MgCl₂, 4 μM tRNA (pH 7.4)] for 10 minutes at 37°C. Then, ligand beacon (SEQ ID NO:2) was added to a final concentrationof 160 nM and the mixture was incubated for 10 minutes at 37° C. Ameasurement of fluorescein emission at 530 nm was made for eachconcentration of PDGF using 488 nm monochromatic laser light forexcitation in a 96 well format Vistra Fluorimager SI. The average valueof fluorescence signals derived from duplicates was plotted against thecorresponding log concentration of the concentration PDGF AB-dimer innanomoles. The results are displayed in FIG. 5A.

Control experiments carried out in the same buffer and temperature, theresults of which are displayed in FIG. 5B. Open circles represent thefluorescence signal obtained when the 100 nM PDGF aptamer was firstincubated with 100 nM PDGF ligand beacon before the protein was added.Closed circles indicate the signal generated when 100 nM PDGF ligandbeacon was mixed with increasing concentration of the PDGF AB-dimer inthe absence of the aptamer.

TAQ DNA Polymerase

An assay using constant concentrations of TAQ nucleic acid ligand andTAQ ligand beacon, and varying concentrations of TAQ DNA polymerase, wascarried out as described above. Again, the fluorescence emissiondecreased with increasing amounts of the ligand Taq Polymerasc. Theresults are shown in FIG. 6.

Human L-Selectin

A fixed concentration of L-selectin aptamer was mixed with increasingconcentration of human L-selectin in SHMCK buffer [20 mM HEPES, 120 mMNaCl. 5 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 4 μM tRNA, (pH 7.4)] andincubated for 10 minutes at 37° C. Then, L-selectin ligand beacon wasadded to a final concentration equivalent to that of the aptamer,incubated additional 10 minutes at 37° C. and fluorescence was measured.The results are depicted in FIGS. 7A and B. It can be seen that thedynamic range of the assay can be easily varied by changing theconcentration of the nucleic acid ligand/ligand beacon pair.

Human P-Selectin

P-selectin ligand beacon (200 nM) was mixed with increasingconcentration of the P-selectin RNA aptamer in SHMCK buffer [20 mMHEPES, 120 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 4 μM tRNA, (pH7.4)] and incubated for 10 minutes at 37° C. The fluorescence signal wasthen measured as described above. The results are depicted in FIG. 8A.

P-selectin aptamer (200 nM) was mixed with increasing concentration ofhuman P-selectin in SHMCK buffer [20 mM HEPES, 120 mM NaCl, 5 mM KCl, 1mM MgCl₂, 1 mM CaCl₂, 4 μM tRNA, (pH 7.4)] and incubated for 10 minutesat 37° C. Then. P-selectin ligand beacon was added to a finalconcentration of 200 nM, incubated for additional 10 minutes at 37° C.and fluorescence was measured. The results are depicted in FIG. 8B.

In order to demonstrate the specificity of the ligand beacon/nucleicacid ligand interaction, an assay was performed in which the P-Selectinnucleic acid ligand was mixed with its cognate ligand beacon andL-Selectin. Specifically, L-Selectin nucleic acid ligand (200 nM) wasmixed with increasing concentrations of P-Selectin in SHMCK buffercontaining 4 μM tRNA. The mixture was incubated at 37° C. for 15minutes. Then 220 nM L-Selectin ligand beacon was added and the mixturewas incubated an additional 10 minutes at the same temperature andfluorescence was measured (FIG. 8C; ()). P-Selectin nucleic acid ligand(200 nM) was mixed with increasing concentrations of L-Selectin in SHMCKbuffer containing 4 μM tRNA at 37° C. for 15 minutes. Then 220 nMP-Selectin ligand beacon was added, incubated for an additional 10minutes at the same temperature and fluorescence was measured (FIG. 8C;(∘)).

As can be seen from the results shown in FIG. 8C, there is a little orno change in the fluorescence intensity when the wrong Selectin isadded. In the presence of the wrong target protein the nucleic acidligand is available for binding to the ligand beacon resulting in highfluorescence. This result indicates that the change in fluorescence isdependent on the presence of the specific target.

Example 6

Use of Ligand Beacons Assays in Plasma

Proteins resuspended in appropriate buffers were added to freshlyprepared plasma such that the plasma concentration was not less than 80%(v/v). To these solutions 10 μL of an aptamer mixed with tRNA in abinding buffer (PBSM or SHMCK) was added such that the finalconcentration of aptamers was either 200 nM or 800 nM (for L-selectin)and the final concentration of tRNA was 4 μM or 16 μM. These reactionmixtures were incubated for 10 minutes at 37° C. Then, 10 μL of ligandbeacon resuspended in an appropriate binding buffer was added to theabove reactions to a final concentration equivalent to the aptamerconcentration. After 10 minutes further incubation at 37° C.,fluorescence was measured. The results are depicted in FIGS. 9A-C, whichdemonstrates that ligand beacons can be successfully used with plasma.

Example 7

Cascade Hybridization

The stem sequences of three stem-loop nucleic acids that willparticipate in a hybridization cascade are represented in FIG. 14A. Theloop of stem-loop I comprises a sequence that is complementary to thetarget nucleic acid sequence that one would like to detect. Upon bindingto the target nucleic acid, then stem of stem-loop I becomes dissociated(FIG. 14B). The arms of the dissociated stem then pair with those ofstem-loop II and stem-loop III (FIG. 14C). The arms of stem-loops II andIII can now pair with those of stem-loops III and It respectively (FIG.14D). This pattern of hybridization between the stems of the threenucleic acids will continue bidirectionally until one of the stem-loopsis depleted.

18 1 38 DNA Artificial Sequence Description of Artificial SequenceSynthetic Sequence 1 tgggagggcg cgttcttcgt ggttactttt agtcccgt 38 2 37DNA Artificial Sequence Description of Artificial Sequence SyntheticSequence 2 gcgagaaagt aaccacgaag aagaacgcgc ccctcgc 37 3 51 DNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 3 tggcggagcg atcatctcag agcattctta gcgttttgtt cttgtgtatg a 51 434 DNA Artificial Sequence Description of Artificial Sequence SyntheticSequence 4 gcgagcaaga acaaaacgct aagaatgctc tcgc 34 5 49 DNA ArtificialSequence Description of Artificial Sequence Synthetic Sequence 5tagccaaggt aaccagtaca aggtgctaaa cgtaatggct tcggcttac 49 6 30 DNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 6 gcgagtgtac tggttacctt ggctactcgc 30 7 38 RNA ArtificialSequence Description of Artificial Sequence Synthetic Sequence 7cucaacgagc caggaacauc gaggucagca aacgcgag 38 8 30 DNA ArtificialSequence Description of Artificial Sequence Synthetic Sequence 8gcgagctcgc gtttgctgac gtcgactcgc 30 9 64 DNA Artificial SequenceDescription of Artificial Sequence Synthetic Sequence 9 ttggtctctggcggagcgat catctcagag cattcttagc gttttgttct tgtgtatgat 60 tcgc 64 10 36DNA Artificial Sequence Description of Artificial Sequence SyntheticSequence 10 ttggagggcg cgttcttcgt ggttactttt agtccc 36 11 49 DNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 11 tagccaaggt aaccagtaca aggtgctaaa cgtaatggct tcggcttac 49 1262 RNA Artificial Sequence Description of Artificial Sequence SyntheticSequence 12 gggagacaag aauaaacgcu caacgagcca ggaacaucga ggucagcaaacgcgagcgcg 60 ag 62 13 10 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 13 aggctagcta 10 14 10 DNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 14 tagggagatt 10 15 10 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 15 aatctcccta 10 16 10 DNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 16 tagtttgagg 10 17 10 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 17 cctcaaacta 10 18 10 DNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 18 tagctagcct 10

What is claimed is:
 1. A method for detecting a target molecule in atest mixture suspected of containing said target molecule, the methodcomprising: A) i) providing a nucleic acid ligand capable of binding tosaid target molecule, said nucleic acid ligand comprising a firstsequence A and a second sequence B wherein A and B are partiallycomplementary sequences that form an imperfect intramolecular duplex,wherein said duplex unwinds upon the binding of said target to saidnucleic acid ligand, and wherein said sequences A and B are able toparticipate in extramolecular hybridization reactions only when saidduplex is unwound; ii) providing a first cascade nucleic acid comprisinga first sequence A′ and a second sequence C′, wherein A′ and C′ arepartially complementary sequences that form an imperfect intramolecularduplex, wherein A′ is exactly complementary to sequence A and whereinthe duplex of said first cascade nucleic acid unwinds upon thehybridization of A′ to an unpaired A sequence; iii) providing a secondcascade nucleic acid, said second cascade nucleic acid comprising afirst sequence B′ and a second sequence C wherein B′ and C are partiallycomplementary sequences that form an imperfect intramolecular duplex,wherein B′ is exactly complementary to sequence B and C is exactlycomplementary to C′, and wherein the duplex of said second cascadenucleic acid unwinds upon the hybridization of B′ to an unpaired Bsequence or upon the hybridization of C′ to an unpaired C sequence; iv)providing a third cascade nucleic acid, said third cascade nucleic acidcomprising a first sequence A and a second sequence B that form animperfect intramolecular duplex and wherein the duplex of said thirdcascade nucleic acid unwinds upon the hybridization of A to A′ or uponthe hybridization of B to B′; B) contacting said test mixture suspectedof containing said target molecule with said nucleic acid ligand,whereby said duplex of said nucleic acid ligand unwinds in the presenceof said target whereby sequences A and B become available forextramolecular hybridization; C) contacting said test mixture and saidnucleic acid ligand with said first, second, and third cascade nucleicacids, wherein the presence of unpaired A and B sequences on saidnucleic acid ligand triggers a cascade of intermolecular hybridizationinvolving said cascade nucleic acids in which intermolecularhybridization takes place between A and A′, between B and B′ and betweenC and C′, leading to the formation of a multimolecular hybridizationcomplex; D) detecting the presence of said multimolecular hybridizationcomplex.
 2. The method of claim 1 wherein at least one of said cascadenucleic acids is labeled at discrete nucleotide positions with at leastone fluorescent group and at least one fluorescence-modifying group,wherein the fluorescence emission profile of each said labeled cascadenucleic acid changes measurably upon unwinding of said intramolecularduplex, wherein said change results from movement of said fluorescencegroups relative to said fluorescence-modifying groups, and wherein stepD is accomplished by monitoring the fluorescence emission profile ofsaid labeled cascade nucleic acid in said target mixture.
 3. The methodof claim 2 wherein each said fluorescence-modifying group is a quenchinggroup, and wherein each said fluorescent group and each said quenchinggroup are located on opposite strands of said intramolecular duplex suchthat fluorescence emission from said fluorescent group is quenched bysaid quenching group, wherein said fluorescent group and said quenchinggroup become spatially separated upon the unwinding of said stem suchthat fluorescence emission from said fluorescence group is no longerquenched by said quenching group.